Therapeutic cd47 antibodies

ABSTRACT

Provided are anti-CD47 monoclonal antibodies (anti-CD47 mAbs) with distinct functional profiles as described herein, methods to generate anti-CD47 mAbs, and to methods of using these anti-CD47 mAbs as therapeutics for the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury, cardiovascular diseases, autoimmune diseases, inflammatory diseases or as diagnostics for determining the level of CD47 in tissue samples.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2017/057716, filed Oct. 20, 2017, which claims the benefit of U.S.Provisional Application No. 62/411,319, filed Oct. 21, 2016, and U.S.Provisional Application No. 62/475,032, filed Mar. 22, 2017, thedisclosures of which are incorporated herein in their entirety.

FIELD OF THE DISCLOSURE

This disclosure is related generally to anti-CD47 monoclonal antibodies(anti-CD47 mAbs) with distinct functional profiles as described herein,methods to generate anti-CD47 mAbs, and methods of using these anti-CD47mAbs as therapeutics for the prevention and treatment of solid andhematological cancers, ischemia-reperfusion injury, cardiovasculardiseases, autoimmune diseases, or inflammatory diseases, or asdiagnostics for determining the level of CD47 in tissue samples.

BACKGROUND OF THE DISCLOSURE

CD47 is a cell surface receptor comprised of an extracellular IgV setdomain, a 5 transmembrane domain, and a cytoplasmic tail that isalternatively spliced. Two ligands bind CD47: signal inhibitory receptorprotein α (SIRPα) and thrombospondin-1 (TSP1). CD47 expression and/oractivity has been implicated in a number of diseases and disorders.Accordingly, there exists a need for therapeutic compositions andmethods for treating diseases and conditions associated with CD47 inhumans and animals, including the prevention and treatment of solid andhematological cancers, ischemia-reperfusion injury (IRI), cardiovasculardiseases, or an autoimmune or inflammatory disease.

SUMMARY OF THE DISCLOSURE

The present disclosure describes anti-CD47 mAbs with distinct functionalprofiles. These antibodies possess distinct combinations of propertiesselected from the following: 1) exhibit cross-reactivity with one ormore species homologs of CD47; 2) block the interaction between CD47 andits ligand SIRPα; 3) increase phagocytosis of human tumor cells; 4)induce death of susceptible human tumor cells; 5) do not induce celldeath of human tumor cells; 6) do not have reduced or minimal binding tohuman red blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) haveminimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10)cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibitionof the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition ofthe NO pathway; 13) cause loss of mitochondrial membrane potential; 14)do not cause cause loss of mitochondrial membrane potential; 15) causean increase in cell surface calreticulin expression on human tumorcells; 16) do not cause an increase in cell surface calreticulinexpression on human tumor cells; 17) cause an increase in adenosinetriphosphate (ATP) release by human tumor cells; 18) do not cause anincrease in adenosine triphosphate (ATP) release by human tumor cells;19) cause an increase in high mobility group box 1 (HMGB1) release byhuman tumor cells; 20) do not cause an increase in high mobility groupbox 1 (HMGB1) release by human tumor cells; 21) cause an increase intype I interferon release by human tumor cells; 22) do not cause anincrease in type I interferon release by human tumor cells; 23) cause anincrease in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by humantumor cells; 24) do not cause an increase in C-X-C Motif ChemokineLigand 10 (CXCL10) release by human tumor cells; 25) cause an increasein cell surface protein disulfide-isomerase A3 (PDIA3) expression onhuman tumor cells; 26) do not cause an increase in cell surface proteindisulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27)cause an increase in cell surface heat shock protein 70 (HSP70)expression on human tumor cells; 28) do not cause an increase in cellsurface heat shock protein 70 (HSP70) expression on human tumor cells;29) cause an increase in cell surface heat shock protein 90 (HSP90)expression on human tumor cells; 30) do not cause an increase in cellsurface heat shock protein 90 (HSP90) expression on human tumor cells;31) have reduced binding to normal human cells, which includes, but isnot limited to, endothelial cells, skeletal muscle cells, epithelialcells, and peripheral blood mononuclear cells (e.g., human aorticendothelial cells, human skeletal muscle cells, human microvascularendothelial cells, human renal tubular epithelial cells, humanperipherial blood CD3+ cells, and human peripheral blood mononuclearcells); 32) do not have reduced binding to normal human cells, whichincludes, but is not limited to, endothelial cells, skeletal musclecells, epithelial cells, and peripheral blood mononuclear cells (e.g.,human aortic endothelial cells, human skeletal muscle cells, humanmicrovascular endothelial cells, human renal tubular epithelial cells,human peripherial blood CD3+ cells, and human peripheral bloodmononuclear cells); 33) have a greater affinity for human CD47 at anacidic pH compared to physiological pH; 34) do not have a greateraffinity for human CD47 at an acidic pH compared to physiological pH;and 35) cause an increase in annexin A1 release by human tumor cells.The anti-CD47 mAbs of the disclosure are useful in various therapeuticmethods for treating diseases and conditions associated with CD47 inhumans and animals, including the prevention and treatment of solid andhematological cancers, autoimmune diseases, inflammatory diseases, IRI,and cardiovascular diseases. The antibodies of the disclosure are alsouseful as diagnostics to determine the level of CD47 expression intissue samples. Embodiments of the disclosure include isolatedantibodies and immunologically active binding fragments thereof;pharmaceutical compositions comprising one or more of the anti-CD47mAbs, preferably chimeric or humanized forms of said anti-CD47 mAbs;methods of therapeutic use of such anti-CD47 monoclonal antibodies; andcell lines that produce these anti-CD47 mAbs.

The embodiments of the disclosure include the mAbs, or antigen-bindingfragments thereof, which are defined herein by reference to specificstructural characteristics, i.e., specified amino acid sequences ofeither the CDRs or entire heavy chain or light chain variable domains.All antibodies of the disclosure bind to CD47.

The monoclonal antibodies, or antigen binding fragments thereof, maycomprise at least one, usually at least three, CDR sequences as providedherein, usually in combination with framework sequences from a humanvariable region or as an isolated CDR peptide. In some embodiments, anantibody comprises at least one light chain comprising the three lightchain CDR sequences provided herein situated in a variable regionframework, which may be, without limitation, a murine or human variableregion framework, and at least one heavy chain comprising the threeheavy chain CDR sequences provided herein situated in a variable regionframework, which may be, without limitation, a human or murine variableregion framework.

Some embodiments of the disclosure are anti-CD47 mAbs, or antigenbinding fragments thereof, comprising a heavy chain variable domaincomprising a variable heavy chain CDR1, variable heavy chain CDR2, and avariable heavy chain CDR3, wherein said variable heavy chain CDR1comprises an amino acid sequence selected from the group consisting of:SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; said variable heavy chainCDR2 comprises an amino acid sequence selected from the group consistingof: SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; and said variable heavychain CDR3 comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10.

The heavy chain variable (V_(H)) domain may comprise any one of thelisted variable heavy chain CDR1 sequences (HCDR1) in combination withany one of the variable heavy chain CDR2 sequences (HCDR2) and any oneof the variable heavy chain CDR3 sequences (HCDR3). However, certainembodiments of HCDR1 and HCDR2 and HCDR3 are are provided that derivefrom a single common V_(H) domain, examples of which are describedherein.

The antibody or antigen binding fragment thereof may additionallycomprise a light chain variable (V_(L)) domain, which is paired with theV_(H) domain to form an antigen binding domain. In some embodiments,light chain variable domains are those comprising a variable light chainCDR1, variable light chain CDR2, and a variable light chain CDR3,wherein said variable light chain CDR1 comprises an amino acid sequenceselected from the group consisting of: SEQ ID NO:11, SEQ ID NO:12, SEQID NO:13, and SEQ ID NO:14; said variable light chain CDR2 optionallycomprises an amino acid sequence selected from the group consisting of:SEQ ID NO:15, SEQ ID NO:16, and SEQ ID NO:17; and said variable lightchain CDR3 optionally comprises an amino acid sequence selected from thegroup consisting of: SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20.

The light chain variable domain may comprise any one of the listedvariable light chain CDR1 sequences (LCDR1) in combination with any oneof the variable light chain CDR2 sequences (LCDR2) and any one of thevariable light chain CDR3 sequences (LCDR3). However, certainembodiments of LCDR1 and LCDR2 and LCDR3 are provided that derive from asingle common V_(L) domain, examples of which are described herein.

Any given CD47 antibody or antigen binding fragment thereof comprising aV_(H) domain paired with a V_(L) domain will comprise a combination of 6CDRs: variable heavy chain CDR1 (HCDR1), variable heavy chain CDR2(HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDR1(LCDR1), variable light chain CDR2 (LCDR2), and variable light chainCDR3 (LCDR3). Although all combinations of 6 CDRs selected from the CDRsequence groups listed above are permissible, and within the scope ofthe disclosure, certain combinations of 6 CDRs are provided.

In some embodiments, combinations of 6 CDRs include, but are not limitedto, the combinations of variable heavy chain CDR1 (HCDR1), variableheavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variablelight chain CDR1 (LCDR1), variable light chain CDR2 (LCDR2), andvariable light chain CDR3 (LCDR3) selected from the group consisting of:

-   -   (i) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:4,        HCDR3 comprising SEQ ID NO:7, LCDR1 comprising SEQ ID NO:11,        LCDR2 comprising SEQ ID NO:15, LCDR3 comprising SEQ ID NO:18;    -   (ii) HCDR1 comprising SEQ ID NO:1, HCDR2 comprising SEQ ID NO:4,        HCDR3 comprising SEQ ID NO:8, LCDR1 comprising SEQ ID NO:11,        LCDR2 comprising SEQ ID NO:15, LCDR3 comprising SEQ ID NO:18;    -   (iii) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID        NO:5, HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID        NO:12, LCDR2 comprising SEQ ID NO:16, LCDR3 comprising SEQ ID        NO:19;    -   (iv) HCDR1 comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5,        HCDR3 comprising SEQ ID NO:9, LCDR1 comprising SEQ ID NO:13,        LCDR2 comprising SEQ ID NO:16, LCDR3 comprising SEQ ID NO:19;        and    -   (v) HCDR1 comprising SEQ ID NO:3, HCDR2 comprising SEQ ID NO:6,        HCDR3 comprising SEQ ID NO:10, LCDR1 comprising SEQ ID NO:14,        LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO:20.

In some embodiments, anti-CD47 mAbs include antibodies or antigenbinding fragments thereof, comprising a heavy chain variable domainhaving an amino acid sequence selected from the group consisting of: theamino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ IDNO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ IDNO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ IDNO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ IDNO:39, and SEQ ID NO:40, and amino acid sequences exhibiting at least90%, 95%, 97%, 98%, or 99% sequence identity to one of the recitedsequences. Alternatively, or in addition, anti-CD47 mAbs includingantibodies or antigen binding fragments thereof, may comprise a lightchain variable domain having an amino acid sequence selected from thegroup consisting of: the amino acid sequences of SEQ ID NO:41, SEQ IDNO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ IDNO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, and SEQID NO:52, and amino acid sequences exhibiting at least 90%, 95%, 97%,98%, or 99% sequence identity to one of the recited sequences.

Although all possible pairing of V_(H) domains and V_(L) domainsselected from the V_(H) and V_(L) domain sequence groups listed aboveare permissible, and within the scope of the disclosure, someembodiments provide certain combinations of V_(H) and V_(L) domains.Accordingly, in some embodiments, anti-CD47 mAbs, or antigen bindingfragments thereof, are those comprising a combination of a heavy chainvariable domain (V_(H)) and a light chain variable domain (V_(L)),wherein the combination is selected from the group consisting of:

-   -   (i) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:21 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:41;    -   (ii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:23 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (iii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:34 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:49;    -   (iv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:36 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (v) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:38 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (vi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:39 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (vii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:24 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (viii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:37 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (ix) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:33 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (x) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:26 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:44;    -   (xi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:27 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:44; and    -   (xii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:38 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51;    -   (xiii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:39 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51;    -   (xiv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:40 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:52;    -   (xv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:36 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51;    -   (xvi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:29 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xvii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:30 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xviii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xix) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:32 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xx) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:33 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:47;    -   (xxi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:29 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:30 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxiii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxiv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:32 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:26 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (xxvi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:27 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:43;    -   (xxvii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:28 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:46;    -   (xxviii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:35 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:50;    -   (xxix) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:29 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxx) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:30 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxxi) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:31 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxxii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:32 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:48;    -   (xxxiii) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:37 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51; and    -   (xxxiv) a heavy chain variable domain comprising the amino acid        sequence of SEQ ID NO:40 and a light chain variable domain        comprising the amino acid sequence SEQ ID NO:51.

In some embodiments, anti-CD47 antibodies or antigen binding fragmentsthereof may also comprise a combination of a heavy chain variable domainand a light chain variable domain wherein the heavy chain variabledomain comprises a V_(H) sequence with at least 85% sequence identity,or at least 90% sequence identity, or at least 95% sequence identity, orat least 97%, 98% or 99% sequence identity, to the heavy chain aminoacid sequences shown above in (i) to (xxxiv) and/or the light chainvariable domain comprises a V_(L) sequence with at least 85% sequenceidentity, or at least 90% sequence identity, or at least 95% sequenceidentity, or at least 97%, 98% or 99% sequence identity, to the lightchain amino acid sequences shown above in (i) to (xxxiv). The specificV_(H) and V_(L) pairings or combinations in parts (i) through (xxxiv)may be preserved for anti-CD47 antibodies having V_(H) and V_(L) domainsequences with a particular percentage sequence identity to thesereference sequences.

For all embodiments wherein the heavy chain and/or light chain variabledomains of the antibodies or antigen binding fragments are defined by aparticular percentage sequence identity to a reference sequence, theV_(H) and/or V_(L) domains may retain identical CDR sequences to thosepresent in the reference sequence such that the variation is presentonly within the framework regions.

In another embodiment, CD47 antibodies, or antigen binding fragmentsthereof, may comprise a combination of a heavy chain (HC) and a lightchain (LC), wherein the combination is selected from the groupconsisting of:

-   -   (i) a heavy chain comprising the amino acid sequence of SEQ ID        NO:78 and a light chain comprising the amino acid sequence SEQ        ID NO:67;    -   (ii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:79 and a light chain comprising the amino acid sequence SEQ        ID NO:69;    -   (iii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:80 and a light chain comprising the amino acid sequence SEQ        ID NO:70;    -   (iv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:81 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (v) a heavy chain comprising the amino acid sequence of SEQ ID        NO:82 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (vi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:83 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (vii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:84 and a light chain comprising the amino acid sequence SEQ        ID NO:69;    -   (viii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:85 and a light chain comprising the amino acid sequence        SEQ ID NO:71;    -   (ix) a heavy chain comprising the amino acid sequence of SEQ ID        NO:86 and a light chain comprising the amino acid sequence SEQ        ID NO:72;    -   (x) a heavy chain comprising the amino acid sequence of SEQ ID        NO:87 and a light chain comprising the amino acid sequence SEQ        ID NO:73;    -   (xi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:88 and a light chain comprising the amino acid sequence SEQ        ID NO:73;    -   (xii) a heavy chain comprising the amino acid sequence of SEQ ID        NO:82 and a light chain comprising the amino acid sequence SEQ        ID NO:74;    -   (xiii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:83 and a light chain comprising the amino acid sequence        SEQ ID NO:74;    -   (xiv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:89 and a light chain comprising the amino acid sequence SEQ        ID NO:71;    -   (xv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:81 and a light chain comprising the amino acid sequence SEQ        ID NO:74;    -   (xvi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:90 and a light chain comprising the amino acid sequence SEQ        ID NO:75;    -   (xvii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:91 and a light chain comprising the amino acid sequence        SEQ ID NO:75;    -   (xviii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:92 and a light chain comprising the amino acid sequence        SEQ ID NO:75;    -   (xix) a heavy chain comprising the amino acid sequence of SEQ ID        NO:93 and a light chain comprising the amino acid sequence SEQ        ID NO:75;    -   (xx) a heavy chain comprising the amino acid sequence of SEQ ID        NO:86 and a light chain comprising the amino acid sequence SEQ        ID NO:75;    -   (xxi) a heavy chain comprising the amino acid sequence of SEQ ID        NO:94 and a light chain comprising the amino acid sequence SEQ        ID NO:72;    -   (xxii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:91 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxiii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:92 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxiv) a heavy chain comprising the amino acid sequence of SEQ        ID NO:93 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxv) a heavy chain comprising the amino acid sequence of SEQ ID        NO:87 and a light chain comprising the amino acid sequence SEQ        ID NO:69;    -   (xxvi) a heavy chain comprising the amino acid sequence of SEQ        ID NO:88 and a light chain comprising the amino acid sequence        SEQ ID NO:69;    -   (xxvii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:95 and a light chain comprising the amino acid sequence        SEQ ID NO:76;    -   (xxviii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:96 and a light chain comprising the amino acid sequence        SEQ ID NO:77;    -   (xxix) a heavy chain comprising the amino acid sequence of SEQ        ID NO:97 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxx) a heavy chain comprising the amino acid sequence of SEQ ID        NO:98 and a light chain comprising the amino acid sequence SEQ        ID NO:72;    -   (xxxi) a heavy chain comprising the amino acid sequence of SEQ        ID NO:99 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxxii) a heavy chain comprising the amino acid sequence of SEQ        ID NO: 100 and a light chain comprising the amino acid sequence        SEQ ID NO:72;    -   (xxxiii) a heavy chain comprising the amino acid sequence of SEQ        ID NO:85 and a light chain comprising the amino acid sequence        SEQ ID NO:74;    -   (xxxiv) a heavy chain comprising the amino acid sequence of SEQ        ID NO:89 and a light chain comprising the amino acid sequence        SEQ ID NO:74;        -   wherein the V_(H) amino acid sequence is at least 90%, 95%,            97%, 98% or 99% identical thereto and the a V_(L) amino acid            sequence is at least 90%, 95%, 97%, 98% or 99% identical            thereto.

In some embodiments, anti-CD47 antibodies as described herein may alsobe characterized by combinations of properties which are not exhibitedby prior art anti-CD47 antibodies proposed for human therapeutic use.Accordingly, in some embodiments, anti-CD47 antibodies described hereinare characterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells; and    -   d. induces death of susceptible human tumor cells.

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. induces death of susceptible human tumor cells; and    -   e. causes no detectable agglutination of human red blood cells        (hRBCs).

In yet another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. induces death of susceptible human tumor cells; and    -   e. causes reduced agglutination of human red blood cells        (hRBCs).

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. induces death of susceptible human tumor cells; and    -   e. has reduced hRBC binding.

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47,    -   b. blocks SIRPα binding to human CD47,    -   c. increases phagocytosis of human tumor cells,    -   d. causes no detectable agglutination of human red blood cells        (hRBCs); and    -   e. has minimal binding to hRBCs.

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. causes no detectable agglutination of human red blood cells        (hRBCs); and    -   e. has reduced hRBC binding.

Additional embodiments of the anti-CD47 antibodies described herein, arealso characterized by combinations of properties which are not exhibitedby prior art anti-CD47 antibodies proposed for human therapeutic use.Accordingly, anti-CD47 antibodies as described herein may be furthercharacterized by one or more among the following characteristics:

-   -   a. causes an increase in cell surface calreticulin expression on        human tumor cells;    -   b. causes an increase in adenosine triphosphate (ATP) release by        human tumor cells;    -   c. causes an increase in high mobility group box 1 (HMGB1)        release by human tumor cells;    -   d. causes an increase in annexin A1 release by human tumor        cells;    -   e. causes an increase in Type I Interferon release by human        tumor cells;    -   f. causes an increase in C-X-C Motif Chemokine Ligand 10        (CXCL10) release by human tumor cells;    -   g. causes an increase in cell surface protein        disulfide-isomerase A3 (PDIA3) expression on human tumor cells;    -   h. causes an increase in cell surface heat shock protein 70        (HSP70) expression on human tumor cells; and    -   i. causes an increase in cell surface heat shock protein 90        (HSP90) expression on human tumor cells.

In another embodiment described herein, the monoclonal antibody, orantigen binding fragment thereof binds to human, non-human primate,mouse, rabbit, and rat CD47.

In yet another embodiment described herein, the monoclonal antibody, orantigen binding fragment thereof specifically also binds to non-humanprimate CD47, wherein non-human primate may include, but is not limitedto, cynomolgus monkey, green monkey, rhesus monkey, and squirrel monkey.

In yet another embodiment described herein, the monoclonal antibody, orantigen binding fragment thereof, has reduced binding to normal humancells, which includes, but is not limited to, endothelial cells,skeletal muscle cells, epithelial cells, and peripheral bloodmononuclear cells (e.g., human aortic endothelial cells, human skeletalmuscle cells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, and humanperipheral blood mononuclear cells).

In yet another embodiment described herein, the monoclonal antibody, orantigen binding fragment thereof, has a greater have a greater affinityfor human CD47 at an acidic pH compared to physiological pH.

In some embodiments, the monoclonal antibody, or antigen bindingfragment thereof, may additionally possess one or more of the followingcharacteristics: 1) exhibit cross-reactivity with one or more specieshomologs of CD47; 2) block the interaction between CD47 and its ligandSIRPα; 3) increase phagocytosis of human tumor cells; 4) induce death ofsusceptible human tumor cells; 5) do not induce cell death of humantumor cells; 6) do not have reduced or minimal binding to human redblood cells (hRBCs); 7) have reduced binding to hRBCs; 8) have minimalbinding to hRBCs; 9) cause reduced agglutination of hRBCs; 10) cause nodetectable agglutination of hRBCs; 11) reverse TSP1 inhibition of thenitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition of the NOpathway; 13) cause loss of mitochondrial membrane potential; 14) do notcause cause loss of mitochondrial membrane potential; 15) cause anincrease in cell surface calreticulin expression on human tumor cells;16) do not cause an increase in cell surface calreticulin expression onhuman tumor cells; 17) cause an increase in adenosine triphosphate (ATP)release by human tumor cells; 18) do not cause an increase in adenosinetriphosphate (ATP) release by human tumor cells; 19) cause an increasein high mobility group box 1 (HMGB1) release by human tumor cells; 20)do not cause an increase in high mobility group box 1 (HMGB1) release byhuman tumor cells; 21) cause an increase in type I interferon release byhuman tumor cells; 22) do not cause an increase in type I interferonrelease by human tumor cells; 23) cause an increase in C-X-C MotifChemokine Ligand 10 (CXCL10) release by human tumor cells; 24) do notcause an increase in C-X-C Motif Chemokine Ligand 10 (CXCL10) release byhuman tumor cells; 25) cause an increase in cell surface proteindisulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26) donot cause an increase in cell surface protein disulfide-isomerase A3(PDIA3) expression on human tumor cells; 27) cause an increase in cellsurface heat shock protein 70 (HSP70) expression on human tumor cells;28) do not cause an increase in cell surface heat shock protein 70(HSP70) expression on human tumor cells; 29) cause an increase in cellsurface heat shock protein 90 (HSP90) expression on human tumor cells;30) do not cause an increase in cell surface heat shock protein 90(HSP90) expression on human tumor cells; 31) have reduced binding tonormal human cells, which includes, but is not limited to, endothelialcells, skeletal muscle cells, epithelial cells, and peripheral bloodmononuclear cells (e.g., human aortic endothelial cells, human skeletalmuscle cells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, and humanperipheral blood mononuclear cells); 32) do not have reduced binding tonormal human cells, which includes, but is not limited to, endothelialcells, skeletal muscle cells, epithelial cells, and peripheral bloodmononuclear cells (e.g., human aortic endothelial cells, human skeletalmuscle cells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, and humanperipheral blood mononuclear cells); 33) have a greater affinity forhuman CD47 at an acidic pH compared to physiological pH; 34) do not havea greater affinity for human CD47 at an acidic pH compared tophysiological pH; and 35) cause an increase in annexin A1 release byhuman tumor cells.

Various forms of the anti-CD47 mAbs disclosed are contemplated herein.For example, the anti-CD47 mAbs can be full-length humanized antibodieswith human frameworks and constant regions of the isotypes, IgA, IgD,IgE, IgG, and IgM, more particularly, IgG1, IgG2, IgG3, IgG4, and insome cases with various mutations to alter Fc receptor function orprevent Fab arm exchange or an antibody fragment, e.g., a F(ab′)2fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc., asdisclosed herein.

In some embodiments, pharmaceutical or veterinary compositions areprovided that comprise one or more of the anti-CD47 mAbs or fragmentsdisclosed herein, optionally chimeric or humanized forms, and apharmaceutically acceptable carrier, diluent, or excipient.

Prior to the present disclosure, there was a need to identify anti-CD47mAbs that possess the functional profiles as described herein. Theanti-CD47 mAbs of the present disclosure exhibit distinct combinationsof properties, particularly combinations of properties that render themAbs particularly advantageous or suitable for use in human therapy,particularly in the prevention and/or treatment of solid andhematological cancers, ischemia-reperfusion injury, autoimmune and/orinflammatory diseases.

In some embodiments, the disclosure provides a monoclonal antibody, oran antigen binding fragment thereof, which: binds to human CD47; blocksSIRPα binding to human CD47; increases phagocytosis of human tumorcells; and induces death of human tumor cells; wherein said monoclonalantibody, or an antigen binding fragment thereof, exhibits pH-dependentbinding to CD47 present on a cell. In other embodiments, the disclosureprovides a monoclonal antibody, or an antigen binding fragment thereof,which: binds to human CD47; blocks SIRPα binding to human CD47;increases phagocytosis of human tumor cells; wherein said monoclonalantibody, or an antigen binding fragment thereof, exhibits pH-dependentbinding to CD47 present on a cell. In other embodiments, the disclosureprovides a monoclonal antibody, or an antigen binding fragment thereof,which: binds to human CD47; blocks SIRPα binding to human CD47;increases phagocytosis of human tumor cells; and induces death of humantumor cells; wherein said monoclonal antibody, or an antigen bindingfragment thereof, exhibits reduced binding to normal cells. In oneembodiment, these cells may be an endothelial cell, a skeletal musclecell, an epithelial cell, a PBMC or a RBC (e.g., human aorticendothelial cells, human skeletal muscle cells, human microvascularendothelial cells, human renal tubular epithelial cells, humanperipherial blood CD3+ cells, human peripheral blood mononuclear cellsor human RBC). In other embodiments, the disclosure provides amonoclonal antibody, or an antigen binding fragment thereof, which:binds to human CD47; blocks SIRPα binding to human CD47; increasesphagocytosis of human tumor cells; wherein said monoclonal antibody, oran antigen binding fragment thereof, exhibits reduced binding to normalcells. In one embodiment, these cells may be an endothelial cell, askeletal muscle cell, an epithelial cell, a PBMC or a RBC (e.g., humanaortic endothelial cells, human skeletal muscle cells, humanmicrovascular endothelial cells, human renal tubular epithelial cells,human peripherial blood CD3+ cells, human peripheral blood mononuclearcells or human RBC). In another embodiment, the monoclonal antibody, oran antigen binding fragment thereof, exhibits both pH dependent bindingand reduced binding to a cell.

Further scope of the applicability of the present disclosure will becomeapparent from the detailed description provided below. However, itshould be understood that the detailed description and specificexamples, while indicating some embodiments of the disclosure, are givenby way of illustration only since various changes and modificationswithin the spirit and scope of the disclosure will become apparent tothose skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other aspects, features, and advantages of the presentdisclosure will be better understood from the following detaileddescriptions taken in conjunction with the accompanying drawing(s), allof which are given by way of illustration only, and are not limitativeof the present disclosure.

FIG. 1A. Binding of VLX4 Humanized mAbs to Human OV10 Cells ExpressingHuman CD47. Binding of VLX4 humanized mAbs (VLX4hum_01 IgG1, VLX4hum_02IgG1, VLX4hum_01 IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 wasdetermined using a OV10 cell line expressing human CD47 (OV10 hCD47)cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates andwere confluent at the time of assay. Various concentrations of mAbs wereadded to the cells for 1 hr. Cells were washed and then incubated withHRP-labelled secondary antibody for 1 hr followed by addition ofperoxidase substrate.

FIG. 1B. Binding of VLX4 Humanized mAbs to Human OV10 Cells ExpressingHuman CD47. Binding of VLX4 humanized mAbs (VLX4hum_06 IgG4PE,VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE) to humanCD47 was determined using an OV10 CD47 cell-based ELISA. OV10 hCD47cells were plated into 96 well plates and were confluent at the time ofassay. Various concentrations of VLX4 representative mAbs were added tothe cells for 1 hr. Cells were washed and then incubated withHRP-labelled secondary antibody for 1 hr followed by addition ofperoxidase substrate.

FIG. 2A. Binding of VLX4 Humanized mAbs to Human RBCs (hRBCs). Bindingof VLX4 humanized mAbs (VLX4hum_01 IgG, VLX4hum_02 IgG, VLX4hum_01IgG4PE, and VLX4hum_02 IgG4PE) to human CD47 was determined usingfreshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37° C.with various concentrations of VLX4 mAbs, washed and incubated for 1 hrwith FITC-labelled donkey anti-human antibody. Cells were washed andantibody binding measured using flow cytometry.

FIG. 2B. Binding of VLX4 Humanized mAbs to Human RBCs. Binding of VLX4humanized mAbs (VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13IgG4PE) to human CD47 was determined using freshly isolated hRBCs. hRBCswere incubated for 60 minutes at 37° C. with various concentrations ofVLX4 mAbs, washed and incubated for 1 hr with FITC-labelled donkeyanti-human antibody. Cells were washed and antibody binding measuredusing flow cytometry.

FIG. 3A. Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX8 IgG4PE chimera (xi) or humanized mAbs (VLX8hum_01IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_09 IgG4PE) tohuman CD47 was determined using an OV10 hCD47 cell-based ELISA. OV10hCD47 cells were plated into 96 well plates and were confluent at thetime of assay. Various concentrations of VLX8 representative mAbs wereadded to the cells for 1 hr. Cells were washed and then incubated withHRP-labelled secondary antibody for 1 hr followed by addition ofperoxidase substrate.

FIG. 3B. Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX8 chimera or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07IgG2, VLX8hum_08 IgG2, and VLX8hum_09 IgG2) to human CD47 was determinedusing an OV10 hCD47 cell-based ELISA. OV10 hCD47 cells were plated into96 well plates and were confluent at the time of assay. Variousconcentrations of VLX8 representative mAbs were added to the cells for 1hr. Cells were washed and then incubated with HRP-labelled secondaryantibody for 1 hr followed by addition of peroxidase substrate.

FIG. 4A. Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8IgG4PE xi or humanized mAbs (VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE,VLX8hum_07 IgG4PE, and VLX8hum_10 IgG4PE) to human CD47 was determinedusing freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C. with various concentrations of VLX8 mAbs, washed and incubated for 1hr with FITC-labelled donkey anti-human antibody. Cells were washed andantibody binding measured using flow cytometry.

FIG. 4B. Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8IgG4PE xi or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2,VLX8hum_08 IgG2 and VLX8hum_09 IgG2) to human CD47 was determined usingfreshly isolated human RBCs. RBCs were incubated for 1 hr at 37° C. withvarious concentrations of VLX8 mAbs, washed and incubated for 1 hr withFITC-labelled donkey anti-human antibody. Cells were washed and antibodybinding measured using flow cytometry.

FIG. 5A. Binding of VLX9 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_01 IgG2, VLX9hum_02IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2 and VLX9hum_05 IgG2) to humanCD47 was determined using an OV10 human CD47 cell-based ELISA. OV10hCD47 cells were plated into 96 well plates and were confluent at thetime of assay. Various concentrations of mAbs were added to the cellsfor 1 hr. Cells were washed and then incubated with HRP-labelledsecondary antibody for 1 hr followed by addition of peroxidasesubstrate.

FIG. 5B. Binding of VLX9 Humanized mAbs to Human OV10 hCD47 Cells.Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_06 IgG2, VLX9hum_07IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) to humanCD47 was determined using a OV10 hCD47 cell-based ELISA. OV10 hCD47cells were plated into 96 well plates and were confluent at the time ofassay. Various concentrations of mAbs were added to the cells for 1 hr.Cells were washed and then incubated with HRP-labelled secondaryantibody for 1 hr followed by addition of peroxidase substrate.

FIG. 6A. Specific Binding of VLX Humanized mAbs to CD47. Binding of VLXhumanized mAb VLX4hum_07 IgG4PE to wildtype and CD47 knockout Jurkatcells was determined by flow cytometry. Various concentrations of mAbswere added to 1×10⁴ cells for 1 hr. The cells were washed and thenincubated with FITC-labelled secondary antibody for 1 hr. Cells werewashed and antibody binding measured using flow cytometry.

FIG. 6B. Specific Binding of VLX Humanized mAbs to CD47. Binding of VLXhumanized mAb VLX9hum_04 IgG2 to wildtype and CD47 knockout Jurkat cellswas determined by flow cytometry. Various concentrations of mAbs wereadded to 1×10⁴ cells for 1 hr. The cells were washed and then incubatedwith FITC-labelled secondary antibody for 1 hr. Cells were washed andantibody binding measured using flow cytometry.

FIG. 7. Binding of VLX9 Humanized mAbs to Human RBCs. Binding of VLX9IgG2 xi or humanized VLX9 mAbs to human CD47 (VLX9hum_01 IgG2,VLX9hum_02 IgG2 and VLX9hum_07 IgG2) was determined using freshlyisolated human hRBCs. RBCs were incubated for 60 minutes at 37° C. withvarious concentrations of VLX9 mAbs, washed and incubated for 1 hr withFITC-labelled donkey anti-human antibody. Cells were washed and antibodybinding measured using flow cytometry.

FIG. 8A. Binding of VLX Humanized mAbs to Human Aortic Endothelial Cells(HAEC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2,VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2)to HAEC was determined by flow cytometry. HAEC were removed from theflask using acutase. Various concentrations of mAbs were added to 1×10⁴cells for 1 hr. The cells were washed and then incubated withFITC-labelled secondary antibody for 1 hr followed by measurement ofFITC label by flow cytometry.

FIG. 8B. Binding of VLX Humanized mAbs to Skeletal Human Muscle Cells(SkMC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2,VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2)to SkMc was determined by flow cytometry. SkMC were removed from theflask using acutase. Various concentrations of mAbs were added to 1×10⁴cells for 1 hr. The cells were washed and then incubated withFITC-labelled secondary antibody for 1 hr followed by measurement ofFITC label by flow cytometry.

FIG. 8C. Binding of VLX Humanized mAbs to Human Lung MicrovascularEndothelial Cells (HMVEC-L). Binding of VLX humanized mAbs (VLX4hum_07IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE,VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 andVLX9hum_04 IgG2) to HMVEC-L was determined by flow cytometry. HMVEC-Lwere removed from the flask using acutase. Various concentrations ofmAbs were added to 1×10⁴ cells for 1 hr. The cells were washed and thenincubated with FITC-labelled secondary antibody for 1 hr followed bymeasurement of FITC label by flow cytometry.

FIG. 8D. Binding of VLX Humanized mAbs to Human Renal Tubular EpithelialCells (RTEC). Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE,VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04IgG2) to RTEC by flow cytometry. RTEC were removed from the flask usingacutase. Various concentrations of mAbs were added to 1×10⁴ cells for 1hr. The cells were washed and then incubated with FITC-labelledsecondary antibody for 1 hr followed by measurement of FITC label byflow cytometry.

FIG. 8E. Binding of VLX Humanized mAbs to Human Peripheral Blood CD3⁺Cells. Binding of VLX humanized mAbs (VLX4hum_07 IgG4PE, VLX8hum_10IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE, VLX9hum_06 IgG2,VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 and VLX9hum_04 IgG2)to CD3⁺ cells was determined by flow cytometry. PBMC were plated into 96well plates. Various concentrations of mAbs were added to the cells for1 hr. Cells were washed and then incubated with FITC-labelled secondaryantibody and (APC)-labelled anti-CD3 antibody for 1 hr followed bymeasurement of FITC-labelled APC-positive cells by flow cytometry.

FIG. 8F. Binding of VLX Humanized mAbs to Human Peripheral BloodMononuclear Cells (PBMC). Binding of VLX humanized mAbs (VLX4hum_07IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE, VLX4hum_01 IgG4PE,VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2, VLX9hum_03 IgG2 andVLX9hum_04 IgG2) to PBMC was determined by flow cytometry. PBMCs wereplated into 96 well plates. Various concentrations of mAbs were added tothe cells for 1 hr. Cells were washed and then incubated withFITC-labelled secondary antibody for 1 hr followed by measurement ofFITC label by flow cytometry.

FIG. 9A. pH Dependent and pH Independent Binding of Humanized mAb toHis-CD47. Binding of VLX9hum_09 IgG2 to human CD47 was determined usinga solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiterwells, washed and various concentrations of humanized mAbs were added tothe wells for 1 hr at pH 6 or 8. The wells were washed and thenincubated with HRP-labelled secondary antibody for 1 hour followed byaddition of peroxidase substrate.

FIG. 9B. pH Dependent and pH Independent Binding of Humanized mAb toHis-CD47. Binding of VLX9hum_04 IgG2 to human CD47 was determined usinga solid-phase CD47 ELISA assay. His-CD47 was adsorbed to microtiterwells, washed and various concentrations of humanized mAbs were added tothe wells for 1 hr at pH 6 or 8. The wells were washed and thenincubated with HRP-labelled secondary antibody for 1 hour followed byaddition of peroxidase substrate.

FIG. 9C. pH Dependent and pH Independent Binding of Humanized mAb toHis-CD47. Binding of VLX4hum_07 IgG4PE to human CD47 was determinedusing a solid-phase CD47 ELISA assay. His-CD47 was adsorbed tomicrotiter wells, washed and various concentrations of humanized mAbswere added to the wells for 1 hr at pH 6 or 8. The wells were washed andthen incubated with HRP-labelled secondary antibody for 1 hour followedby addition of peroxidase substrate.

FIG. 9D. pH Dependent and pH Independent Binding of Humanized mAb toHis-CD47. Binding of VLX8hum_10 IgG4PE to human CD47 was determinedusing a solid-phase CD47 ELISA assay. His-CD47 was adsorbed tomicrotiter wells, washed and various concentrations of humanized mAbswere added to the wells for 1 hr at pH 6 or 8. The wells were washed andthen incubated with HRP-labelled secondary antibody for 1 hour followedby addition of peroxidase substrate.

FIG. 10. VLX4, VLX8, and VLX9 Humanized mAbs Block SIRPα binding to CD47on Human Jurkat Cells. 1.5×10⁶ Jurkat cells were incubated with 5 μg/mlof VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX4hum_11 IgG4PE, VLX9hum_03IgG2, VLX9hum_06 IgG2, and VLX9hum_08 IgG2) or a control antibody or noantibody in RPMI containing 10% FBS for 30 min at 37° C. An equal volumeof media containing fluorescently labelled SIRPα-Fc fusion protein wasadded and incubated for an additional 30 min at 37° C. Cells were washedand binding was assessed using flow cytometry.

FIG. 11. VLX4 CD47 Chimeric mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hrs. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/ml ofthe VLX4 chimeric mAbs (VLX4 IgG1 xi, VLX4 IgG1 N297Q xi, VLX4 IgG4PExi, VLX4 IgG4 S228P xi) were added to the macrophage cultures andincubated at 37° C. for 2 hrs. Non-phagocytosed Jurkat cells wereremoved and macrophage cultures were washed extensively. Macrophageswere trypsinized and stained for CD14. Flow cytometry was used todetermine the percentage of CD14⁺/CFSE⁺ cells in the total CD14⁺population.

FIG. 12A. VLX4 Humanized mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hrs. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/ml ofantibody (VLX4hum_01 IgG1 and VLX4hum_01 IgG4PE) were added to themacrophage cultures and incubated at 37° C. for 2 hrs. Non-phagocytosedJurkat cells were removed and macrophage cultures were washedextensively. Macrophages were trypsinized and stained for CD14. Flowcytometry was used to determine the percentage of CD14⁺/CFSE⁺ cells inthe total CD14⁺ population.

FIG. 12B. VLX4 Humanized mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hrs. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/ml ofantibody (VLX4 IgG4PE xi, VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE,VLX4hum_012 IgG4PE and VLX4hum_13 IgG4PE) were added to the macrophagecultures and incubated at 37° C. for 2 hrs. Non-phagocytosed Jurkatcells were removed and macrophage cultures were washed extensively.Macrophages were trypsinized and stained for CD14. Flow cytometry wasused to determine the percentage of CD14⁺/CFSE⁺ cells in the total CD14⁺population.

FIG. 13A. VLX8 CD47 Chimeric mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hrs. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/ml ofthe VLX8 chimeric mAbs (VLX8 IgG1 N297Q xi and VLX8 Ig4PE xi) were addedto the macrophage cultures and incubated at 37° C. for 2 hrs.Non-phagocytosed Jurkat cells were removed and macrophage cultures werewashed extensively. Macrophages were trypsinized and stained for CD14.Flow cytometry was used to determine the percentage of CD14⁺/CFSE⁺ cellsin the total CD14⁺ population.

FIG. 13B. VLX8 Humanized mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hrs. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/ml ofantibody (VLX8 IgG4PE xi, VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE,VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE and VLX8hum_09 IgG4PE) were addedto the macrophage cultures and incubated at 37° C. for 2 hrs.Non-phagocytosed Jurkat cells were removed and macrophage cultures werewashed extensively. Macrophages were trypsinized and stained for CD14.Flow cytometry was used to determine the percentage of CD14⁺/CFSE⁺ cellsin the total CD14⁺ population.

FIG. 14A. VLX9 CD47 Chimeric mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hours. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/mlof the VLX9 chimeric mAbs (VLX9 IgG1 N297 xi, VLX9 IgG2 xi and VLX9IgG4PE xi) were added to the macrophage cultures and incubated at 37° C.for two hours. Non-phagocytosed Jurkat cells were removed and macrophagecultures were washed extensively. Macrophages were trypsinized andstained for CD14. Flow cytometry was used to determine the percentage ofCD14+/CFSE+ cells in the total CD14+ population.

FIG. 14B. VLX9 Humanized mAbs Increase Phagocytosis of Human JurkatCells by Human Macrophages. Human macrophages were plated at aconcentration of 1×10⁴ cells per well in a 96 well plate and allowed toadhere for 24 hours. 5×10⁴ CFSE-labelled human Jurkat cells and 1 μg/mlof antibody (VLX9 IgG2 xi, VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2, VLX9hum_06 IgG2, VLX9hum_07IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) were addedto the macrophage cultures and incubated at 37° C. for two hours.Non-phagocytosed Jurkat cells were removed and macrophage cultures werewashed extensively. Macrophages were trypsinized and stained for CD14.Flow cytometry was used to determine the percentage of CD14+/CFSE+ cellsin the total CD14+ population.

FIG. 15A. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX4humanized mAbs (VLX4hum_01 IgG, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1,VLX4hum_02 IgG4PE) in RPMI media for 24 hours at 37° C. Cells were thenstained with annexin V and the signal was detected by flow cytometry.The data are shown as % of cells that are annexin V positive (annexinV+).

FIG. 15B. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX4humanized mAbs (VLX4hum_01 IgG, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1,VLX4hum_02 IgG4PE) in RPMI media for 24 hours at 37° C. Cells were thenstained with annexin V and 7-AAD and analyzed by flow cytometry. Thedata are shown as % of the cells that are annexin V positive/7-AADnegative (annexin V⁺/7-AAD⁻).

FIG. 15C. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX4humanized mAbs (VLX4hum_01 IgG, VLX4hum_01 IgG4PE, VLX4hum_02 IgG1,VLX4hum_02 IgG4PE) in RPMI media for 24 hours at 37° C. Cells were thenstained with annexin V and 7-AAD and analyzed by flow cytometry. Thedata are shown as % of cells that are annexin V positive/7-AAD positive(annexin V⁺/7-AAD⁺).

FIG. 15D. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX4humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE,VLX4hum_11 IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI mediafor 24 hours at 37° C. Cells were then stained with annexin V and 7-AADand analyzed by flow cytometry. The data are shown as the % of cellsthat are annexin V positive (annexin V+).

FIG. 15E. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX4humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE,VLX4hum_11 IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI mediafor 24 hours at 37° C. Cells were then stained with annexin V and 7-AADby flow cytometry. The data are shown as the % of cells that are annexinV positive/7-AAD negative (annexin V⁺/7-AAD⁻).

FIG. 15F. Induction of Cell Death in Human Jurkat Cells by Soluble VLX4Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX4humanized mAbs (VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE, VLX4hum_08 IgG4PE,VLX4hum_11 IgG4PE, VLX4hum_12 IgG4PE, VLX4hum_13 IgG4PE) in RPMI mediafor 24 hours at 37° C. Cells were then stained with annexin V and and7-AAD and analyzed by flow cytometry. The data are shown as the % ofcells that are annexin V positive/7-AAD positive (annexin⁺/7-AAD⁺).

FIG. 16A. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8CD47 Chimeric mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/mlVLX8 chimeric mAbs (VLX8 IgG1 N297Q xi and VLX8 IgG4PE xi) in RPMI mediafor 24 hrs at 37° C. Cells were then stained with annexin V and analyzedby flow cytometry. The data are presented as % of cells that are annexinV positive (annexin V+).

FIG. 16B. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8Chimeric mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX8chimeric mAbs (VLX8 IgG1 N297Q xi and VLX8 IgG4PE xi) in RPMI media for24 hrs at 37° C. Cells were then stained with annexin V and 7-AAD andanalyzed by flow cytometry. The data are presented as the % of cellsthat are annexin V positive/7-AAD negative (annexin V⁺/7-AAD⁻).

FIG. 16C. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8Chimeric mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX8chimeric mAbs (VLX8 IgG1 N297Q xi and VLX8 IgG4PE (xi) in RPMI media for24 hrs at 37° C. Cells were then stained with annexin V and 7-AAD andanalyzed by flow cytometry. The data are presented as the % of cellsthat are annexin V positive/7-AAD positive (annexin V⁺/7-AAD⁺).

FIG. 16D. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX8humanized mAbs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PEand VLX8hum_08 IgG4PE) and chimeric mAb VLX8 IgG4PE in RPMI media for 24hrs at 37° C. Cells were then stained with annexin V and analyzed byflow cytometry. The data are presented as the % of cells that areannexin V positive (annexin V+).

FIG. 16E. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX8humanized mAbs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PEand VLX8hum_08 IgG4PE) and chimeric mAb VLX8 IgG4PE in RPMI media for 24hrs at 37° C. Cells were then stained with annexin V and 7-AAD andanalyzed by flow cytometry. The data are shown as the % of cells thatare annexin V positive/7-AAD negative (annexin V⁺/7-AAD⁻).

FIG. 16F. Induction of Cell Death in Human Jurkat Cells by Soluble VLX8Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX8humanized mAbs (VLX8hum_02 IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_07 IgG4PEand VLX8hum_08 IgG4PE) and chimeric mAb VLX8 IgG4PE in RPMI media for 24hrs at 37° C. Cells were then stained with annexin V and 7-AAD andanalyzed by flow cytometry. The data are shown as the % of cells thatare annexin V positive/7-AAD positive (annexin V⁺/7-AAD⁺).

FIG. 17A. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9Chimeric mAbs. 1×10⁴ Jurkat cells were incubated with 1 μg/ml of theVLX9 CD47 chimeric mAbs (VLX9 IgG1 N297Q xi, VLX9 IgG2 xi and VLX9IgG4PE xi) in RPMI media for 24 hours 37° C. Cells were then stainedwith annexin V and the signal analyzed by flow cytometry. The data areshown as % of cells that are annexin V positive (annexin V+).

FIG. 17B. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9Chimeric mAbs. 1×10⁴ Jurkat cells were incubated with 1 μg/ml of theVLX9 CD47 chimeric mAbs (VLX9 IgG1 N297Q xi, VLX9 IgG2 xi and VLX9IgG4PE xi) in RPMI media for 24 hours 37° C. Cells were then stainedwith annexin V and 7-AAD and analyzed by flow cytometry. The data areshown as % of cells that are annexin V positive/7-AAD negative (annexinV⁺/7-AAD⁻).

FIG. 17C. Induction of Cell Death of Human Jurkat Cells by Soluble VLX9Chimeric mAbs. 1×10⁴ Jurkat cells were incubated with 1 μg/ml of theVLX9 CD47 chimeric mAbs (VLX9 IgG1 N297Q xi, VLX9 IgG2 xi and VLX9IgG4PE xi) in RPMI media for 24 hours 37° C. Cells were then stainedwith annexin V and 7-AAD and analyzed by flow cytometry. The data areshown as % of cells that are annexin V positive/7-AAD positive (annexinV⁺/7-AAD⁺).

FIG. 17D. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX9humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAb VLX9 IgG2 xi inRPMI media for 24 hours at 37° C. Cells were then stained with annexin Vand the signal was detected by flow cytometry. VLX9 IgG2 (xi) is amurine/human chimera. The data are shown as % of cells that are annexinV positive (annexin V⁺).

FIG. 17E. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX9humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAb VLX9 IgG2 xi inRPMI media for 24 hours at 37° C. Cells were then stained with annexin Vand 7-AAD and analyzed by flow cytometry. VLX9 IgG2 (xi) is amurine/human chimera. The data are shown as % of cells that are annexinV positive/7-AAD negative (annexin V⁺/7-AAD⁻).

FIG. 17F. Induction of Cell Death in Human Jurkat Cells by Soluble VLX9Humanized mAbs. Jurkat cells (1×10⁴) were incubated with 1 μg/ml VLX9humanized mAbs (VLX9hum_01 to 10 IgG2) and chimeric mAb VLX9 IgG2 xi inRPMI media for 24 hours at 37° C. Cells were then stained with annexin Vand 7-AAD and analyzed by flow cytometry. VLX9 IgG2 (xi) is amurine/human chimera. The data are shown as the % of cells that areannexin V positive/7-AAD positive (annexin V⁺/7-AAD⁺).

FIG. 18. Induction of Mitochondrial Depolarization in Human Raji Cellsby Soluble VLX4, VLX8 and VLX9 Humanized mAbs. 1×10⁴ Raji cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and the change in JC-1 dyefluorescence was assessed using flow cytometry. The data are expressedas % of cells with mitochondrial depolarization.

FIG. 19. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Cause an Increase inCell Surface Calreticulin Expression on Human Raji Cells. 1×10⁴ Rajicells were incubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanizedmAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgGcontrol antibody or 1 μM of mitoxantrone as a positive control in RPMImedia at 37° C. for 24 hours. Cells were washed and calreticulinexpression was assessed using flow cytometry. The data are expressed as% of cells that are calreticulin positive.

FIG. 20. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Cause an Increase inCell Surface Protein Disulfide-Isomerase A3 (PDIA3) Expression on HumanRaji Cells. 1×10⁴ Raji cells were incubated with 10 μg/ml of VLX4, VLX8and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_l1 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03IgG2), a negative IgG control antibody or 1 μM of mitoxantrone as apositive control in RPMI media at 37° C. for 24 hours. Cells were washedand PDIA3 expression was assessed using flow cytometry. The data areexpressed as % of cells that are PDIA3 positive.

FIG. 21. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CellSurface HSP70 Expression on Human Raji Cells. 1×10⁴ Raji cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and HSP70 expression was assessedusing flow cytometry. The data are expressed as % of cells that areHSP70 positive.

FIG. 22. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CellSurface HSP90 Expression on Human Raji Cells. 1×10⁴ Raji cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and HSP90 expression was assessedusing flow cytometry. The data are expressed as % of cells that areHSP90 positive.

FIG. 23. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase Release ofAdenosine Triphosphate (ATP) by Human Raji Cells. 1×10⁴ Raji cells wereincubated with μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cell-free supernatant was collected and analyzedusing an ATP determination kit. The data are expressed as pM ATP in thesupernatant.

FIG. 24. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Cause an Increase inRelease of High Mobility Group Box 1 (HMGB1) by Human Raji Cells. 1×10⁴Raji cells were incubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2), a negative IgGcontrol antibody or 1 μM of mitoxantrone as a positive control in RPMImedia at 37° C. for 24 hours. Cell-free supernatant was collected andanalyzed using an HMGB1 immunoassay. The data are expressed as ng/ml ofHMGB1 in the supernatant.

FIG. 25. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CXCL10Release by Human Raji Cells. 1×10⁴ Raji cells were incubated with 10μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2and VLX9hum_08 IgG2), a negative IgG control antibody or 1 μM ofmitoxantrone as a positive control in RPMI media at 37° C. for 24 hours.Cell-free supernatant was collected and analyzed using an CXCL10immunoassay. The data are expressed as pg/ml of CXCL10 in thesupernatant.

FIG. 26. Induction Mitochondrial Depolarization in Human Jurkat Cells bySoluble VLX4, VLX8 and VLX9 Humanized mAbs. 1×10⁴ Jurkat cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and the change in JC-1 dyefluorescence was assessed using flow cytometry. The data are expressedas % of cells with mitochondrial depolarization.

FIG. 27. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CellSurface Calreticulin Expression on Human Jurkat Cells. 1×10⁴ Jurkatcells were incubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanizedmAbs (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgGcontrol antibody or 1 μM of mitoxantrone as a positive control in RPMImedia at 37° C. for 24 hours. Cells were washed and calreticulinexpression was assessed using flow cytometry. The data are expressed as% of cells that are calreticulin positive.

FIG. 28. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CellSurface PDIA3 Expression on Human Jurkat Cells. 1×10⁴ Jurkat cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and PDIA3 expression was assessedusing flow cytometry. The data are expressed as % of cells that arePDIA3 positive.

FIG. 29. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CellSurface HSP70 Expression on Human Jurkat Cells. 1×10⁴ Jurkat cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and HSP70 expression was assessedusing flow cytometry. The data are expressed as % of cells that areHSP70 positive.

FIG. 30. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase CellSurface HSP90 Expression on Human Jurkat Cells. 1×10⁴ Jurkat cells wereincubated with 10 μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs(VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2), a negative IgG controlantibody or 1 μM of mitoxantrone as a positive control in RPMI media at37° C. for 24 hours. Cells were washed and HSP90 expression was assessedusing flow cytometry. The data are expressed as % of cells that areHSP90 positive.

FIG. 31. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase ATP Releaseby Human Jurkat Cells. 1×10⁴ Jurkat cells were incubated with 10 μg/mlof VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2and VLX9hum_03 IgG2), a negative IgG control antibody or 1 μM ofmitoxantrone as a positive control in RPMI media at 37° C. for 24 hours.Cell-free supernatant was collected and analyzed using an ATPdetermination kit. The data are expressed as pM ATP in the supernatant.

FIG. 32. Soluble VLX4, VLX8 and VLX9 Humanized mAbs Increase HMGB1Release by Human Jurkat Cells. 1×10⁴ Jurkat cells were incubated with 10μg/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4PE,VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2and VLX9hum_08 IgG2), a negative IgG control antibody or 1 μM ofmitoxantrone as a positive control in RPMI media at 37° C. for 24 hours.Cell-free supernatant was collected and analyzed using an HMGB1immunoassay. The data are expressed as ng/ml of HMGB1 in thesupernatant.

FIG. 33A. Agglutination of hRBCs by VLX4 Humanized mAbs.Hemagglutination was assessed following incubation of hRBCs with variousconcentrations of humanized VLX4 mAbs (VLX4hum_01 IgG1 and VLX4hum_01IgG4PE). Blood was diluted (1:50) and washed 3 times with PBS/EDTA/BSA.hRBCs were added to U-bottomed 96 well plates with equal volumes of theantibodies (75 μl) and incubated for 3 hrs at 37° C. and overnight at 4°C.

FIG. 33B. Agglutination of hRBCs by VLX8 Chimeric and Humanized mAbs.Hemagglutination was assessed following incubation of hRBCs with variousconcentrations of humanized VLX8 mAbs (VLX8hum_01 IgG4PE, VLX8hum_02IgG4PE VLX8hum_03 IgG4PE, VLX8hum_08 IgG4PE, VLX8hum_09 IgG4PE,VLX8hum_10 IgG4PE and VLX8hum_11 IgG4PE) and the chimeric mAb VLX8IgG4PE xi. Blood was diluted (1:50) and washed 3 times withPBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equalvolumes of the antibodies (75 μl) and incubated for 3 hrs at 37° C. andovernight at 4° C.

FIG. 34A. Agglutination of Human RBCs by VLX9 Humanized mAbs.Hemagglutination was assessed following incubation of human RBCs withvarious concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs(VLX9hum_01 to 05 IgG2). Blood was diluted (1:50) and washed 3 timeswith PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates withequal volumes of the antibodies (75 μl) and incubated for 3 hrs at 37°C. and overnight at 4° C.

FIG. 34B. Agglutination of Human RBCs by VLX9 Humanized mAbs.Hemagglutination was assessed following incubation of human RBCs withvarious concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs(VLX9hum_06 to _10 IgG2). Blood was diluted (1:50) and washed 3 timeswith PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates withequal volumes of the antibodies (75 μl) and incubated for 3 hrs at 37°C. and overnight at 4° C.

FIG. 35. VLX4 Humanized mAb Reduces Tumor Growth in Raji XenograftModel. Female NSG mice were inoculated subcutaneously in the right flankwith 0.1 mL of a 30% RPMI/70% Matrigel™ mixture containing a suspensionof 5×10⁶ Raji tumor cells. Five days following inoculation, tumorvolumes were measured and mice with palpable tumor volumes of 31-74 mm³were randomized into 8-10/group. VLX4hum_07 IgG4PE or PBS (control)administration was initiated at this time. Mice were treated with 5mg/kg of antibody 5×/week for 4 weeks by intraperitoneal injection.Tumor volumes and body weights were recorded twice weekly.

FIG. 36. VLX8 Humanized mAb Reduces Tumor Growth in Raji XenograftModel. Female NSG mice were inoculated subcutaneously in the right flankwith 0.1 mL of a 30% RPMI/70% Matrigel™ mixture containing a suspensionof 5×10⁶ Raji tumor cells. Five days following inoculation, tumorvolumes were measured and mice with palpable tumor volumes of 31-74 mm³were randomized into 8-10/group. VLX8hum_10 IgG4PE or PBS (control)administration was initiated at this time. Mice were treated with 5mg/kg of antibody 5×/week for 4 weeks by intraperitoneal injection.Tumor volumes and body weights were recorded twice weekly.

FIG. 37. VLX9 Humanized mAb Reduces Tumor Growth in Raji XenograftModel. Female NSG mice were inoculated subcutaneously in the right flankwith 0.1 mL of a 30% RPMI/70% Matrigel™ mixture containing a suspensionof 5×10⁶ Raji tumor cells. Five days following inoculation, tumorvolumes were measured and mice with palpable tumor volumes of 31-74 mm³were randomized into 8-10/group. VLX9hum_08 IgG2 or PBS (control)administration was initiated at this time. Mice were treated with 5mg/kg of antibody 5×/week for 4 weeks by intraperitoneal injection.Tumor volumes and body weights were recorded twice weekly.

FIG. 38A. Hemoglobin Levels in Blood Following Administration of aHumanized VLX9 mAb to Cynomolgus Monkeys by Intravenous Infusion.VLX9hum_08 IgG2 or vehicle were administered as a one hour intravenousinfusion a dose of 5 mg/kg on day 1 and a dose of 15 mg/kg on day 18.Hemoglobin levels were monitored throughout the study and normalized tocontrol values.

FIG. 38B. RBC Levels in Blood Following Administration of Humanized VLX9mAbs to Cynomolgus Monkeys by Intravenous Infusion. VLX9hum_08 IgG2 orvehicle was administered as a one hour intraveneous infusion a dose of 5mg/kg on day 1 and a dose of 15 mg/kg on day 18. RBC levels weremonitored throughout the study and normalized to control values.

DETAILED DESCRIPTION OF THE DISCLOSURE Definitions

Unless otherwise defined, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures utilized in connection with, and techniques of, cell andtissue culture, molecular biology, and protein and oligo orpolynucleotide chemistry and hybridization described herein are thosewell-known and commonly used in the art.

As used herein, the term “CD47,” “integrin-associated protein (IAP),”“ovarian cancer antigen OA3,” “Rh-related antigen,” and “MERG” aresynonymous and may be used interchangeably.

The term “anti-CD47 antibody” refer to an antibody of the disclosurewhich is intended for use as a therapeutic or diagnostic agent, andtherefore will typically possess the binding affinity required to beuseful as a therapeutic and/or diagnostic agent.

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” or “directed against” is meant that the antibodyreacts with one or more antigenic determinants of the desired antigenand does not react with other polypeptides or binds at a much loweraffinity (Kd>10⁻⁶). Antibodies include but are not limited to,polyclonal, monoclonal, chimeric, Fab fragments, Fab′ fragments, F(ab′)2fragments, single chain Fv fragments, and one-armed antibodies.

As used herein, the term “monoclonal antibody” (mAb) as applied to thepresent antibody compounds refers to an antibody that is derived from asingle copy or clone including, for example, any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.mAbs of the present disclosure preferably exist in a homogeneous orsubstantially homogeneous population. Complete mAbs contain 2 heavychains and 2 light chains.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); and multispecific antibodies formed from antibodyfragments.

As disclosed herein, “antibody compounds” refers to mAbs andantigen-binding fragments thereof. Additional antibody compoundsexhibiting similar functional properties according to the presentdisclosure can be generated by conventional methods. For example, micecan be immunized with human CD47 or fragments thereof, the resultingantibodies can be recovered and purified, and determination of whetherthey possess binding and functional properties similar to or the same asthe antibody compounds disclosed herein can be assessed by the methodsdescribed in Examples 3-17 below. Antigen-binding fragments can also beprepared by conventional methods. Methods for producing and purifyingantibodies and antigen-binding fragments are well known in the art andcan be found, for example, in Harlow and Lane (1988) Antibodies, ALaboratory Manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., chapters 5-8 and 15.

The monoclonal antibodies encompass antibodies in which a portion of theheavy and/or light chain is identical with, or homologous to,corresponding sequences in murine antibodies, in particular the murineCDRs, while the remainder of the chain(s) is (are) identical with, orhomologous to, corresponding sequences in human antibodies. Otherembodiments of the disclosure include antigen-binding fragments of thesemonoclonal antibodies that exhibit binding and biological propertiessimilar or identical to the monoclonal antibodies. The antibodies of thepresent disclosure can comprise kappa or lambda light chain constantregions, and heavy chain IgA, IgD, IgE, IgG, or IgM constant regions,including those of IgG subclasses IgG1, IgG2, IgG3, and IgG4 and in somecases with various mutations to alter Fc receptor function.

The monoclonal antibodies containing the presently disclosed murine CDRscan be prepared by any of the various methods known to those skilled inthe art, including recombinant DNA methods.

Reviews of current methods for antibody engineering and improvement canbe found, for example, in P. Chames, Ed., (2012) Antibody Engineering:Methods and Protocols, Second Edition (Methods in Molecular Biology,Book 907), Humana Press, ISBN-10: 1617799734; C. R. Wood, Ed., (2011)Antibody Drug Discovery (Molecular Medicine and Medicinal Chemistry,Book 4), Imperial College Press; R. Kontermann and S. Dubel, Eds.,(2010) Antibody Engineering Volumes 1 and 2 (Springer Protocols), SecondEdition; and W. Strohl and L. Strohl (2012) Therapeutic antibodyengineering: Current and future advances driving the strongest growtharea in the pharmaceutical industry, Woodhead Publishing.

Methods for producing and purifying antibodies and antigen-bindingfragments are well known in the art and can be found, for example, inHarlow and Lane (1988) Antibodies, A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 5-8 and 15.

A full-length antibody as it exists naturally is a “Y” shapedimmunoglobulin (Ig) molecule comprising four polypeptide chains: twoidentical heavy (H) chains and two identical light (L) chains,interconnected by disulfide bonds. The amino terminal portion of eachchain, termed the fragment antigen binding region (FAB), includes avariable region of about 100-110 or more amino acids primarilyresponsible for antigen recognition via the complementarity determiningregions (CDRs) contained therein. The carboxy-terminal portion of eachchain defines a constant region (the “Fc” region) primarily responsiblefor effector function.

The CDRs are interspersed with regions that are more conserved, termedframeworks (“FRs”). Amino acid sequences of many FRs are well known inthe art. Each light chain variable region (LCVR) and heavy chainvariable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4. The 3 CDRs of the light chain are referred toas “LCDR1, LCDR2, and LCDR3” and the 3 CDRs of the heavy chain arereferred to as “HCDR1, HCDR2, and HCDR3.” The CDRs contain most of theresidues which form specific interactions with the antigen. Thenumbering and positioning of CDR amino acid residues within the LCVR andHCVR regions are in accordance with the well-known Kabat numberingconvention Kabat et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242.

As described herein, the “antigen-binding site” can also be defined asthe “hypervariable regions,” “HVRs,” or “HVs,” and refer to thestructurally hypervariable regions of antibody variable domains asdefined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917,1987). There are six HVRs, three in VH (H1, H2, H3) and three in VL (L1,L2, L3). The CDRs used herein are as defined by Kabat except in H-CDR1,which is extended to include H1.

There are five types of mammalian immunoglobulin (Ig) heavy chains,denoted by the Greek letters α (alpha), δ (delta), ε (epsilon), γ(gamma), and μ (mu), which define the class or isotype of an antibody asIgA, IgD, IgE, IgG, or IgM, respectively. IgG antibodies can be furtherdivided into subclasses, for example, IgG1, IgG2, IgG3, and IgG4.

Each heavy chain type is characterized by a particular constant regionwith a sequence well known in the art. The constant region is identicalin all antibodies of the same isotype, but differs in antibodies ofdifferent isotypes. Heavy chains γ, μ, and δ have a constant regioncomposed of three tandem immunoglobulin (Ig) domains, and a hinge regionfor added flexibility. Heavy chains t and E have a constant regioncomposed of four Ig domains.

The hinge region is a flexible amino acid stretch that links the Fc andFab portions of an antibody. This regions contains cysteine residuesthat can form disulfide bonds, connecting two heavy chains together.

The variable region of the heavy chain differs in antibodies produced bydifferent B cells, but is the same for all antibodies produced by asingle B cell or B cell clone. The variable region of each heavy chainis approximately 110 amino acids long and is composed of a single Igdomain.

In mammals, light chains are classified as kappa (κ) or lambda (λ), andare characterized by a particular constant region as known in the art. Alight chain has two successive domains: one variable domain at theamino-terminal end, and one constant domain at the carboxy-terminal end.Each antibody contains two light chains that are always identical; onlyone type of light chain, κ or λ, is present per antibody in mammals.

The Fc region, composed of two heavy chains that contribute three orfour constant domains depending on the class of the antibody, plays arole in modulating immune cell activity. By binding to specificproteins, the Fc region ensures that each antibody generates anappropriate immune response for a given antigen. The Fc region alsobinds to various cell receptors, such as Fc receptors, and other immunemolecules, such as complement proteins. By doing this, it mediatesdifferent physiological effects, including opsonization, cell lysis, anddegranulation of mast cells, basophils and eosinophils.

As used herein, the term “epitope” refers to a specific arrangement ofamino acids located on a peptide or protein to which an antibody orantibody fragment binds. Epitopes often consist of a chemically activesurface grouping of molecules such as amino acids or sugar side chains,and have specific three dimensional structural characteristics as wellas specific charge characteristics. Epitopes can be linear, i.e.,involving binding to a single sequence of amino acids, orconformational, i.e., involving binding to two or more sequences ofamino acids in various regions of the antigen that may not necessarilybe contiguous in the linear sequence.

As used herein, the terms “specifically binds,” “bind specifically,”“specific binding,” and the like as applied to the present antibodycompounds refer to the ability of a specific binding agent (such as anantibody) to bind to a target molecular species in preference to bindingto other molecular species with which the specific binding agent andtarget molecular species are admixed. A specific binding agent is saidspecifically to recognize a target molecular species when it can bindspecifically to that target.

As used herein, the term “binding affinity” refers to the strength ofbinding of one molecule to another at a site on the molecule. If aparticular molecule will bind to or specifically associate with anotherparticular molecule, these two molecules are said to exhibit bindingaffinity for each other. Binding affinity is related to the associationconstant and dissociation constant for a pair of molecules as measuredin a 1:1 interaction. Affinities as used herein to describe interactionsbetween molecules of the described methods which can be used to comparethe relative strength with which one molecule (e.g., an antibody orother specific binding partner) will bind two other molecules (e.g., twoversions or variants of a peptide) in a univalent interaction. Theconcepts of binding affinity, association constant, and dissociationconstant are well known.

As used herein, the term “apparent binding affinity” refers to theapparent strength of binding of one molecule to another at a site on themolecule. If a particular molecule will bind to or specificallyassociate with another particular molecule, these two molecules are saidto exhibit binding affinity for each other. Apparent binding affinity isrelated to the association constant and dissociation constant for a pairof molecules, and relates to a non 1:1 or multivalent associationbetween the pair of molecules. Apparent affinities as used herein todescribe interactions between molecules of the described methods areobserved in empirical studies, which can be used to compare the relativestrength with which one molecule (e.g., an antibody or other specificbinding partner) will bind two other molecules (e.g., two versions orvariants of a peptide). The concept of binding affinity may be describedas apparent Kd, apparent binding constant, EC₅₀ or other measurements ofbinding.

As used herein, the term “sequence identity” means the percentage ofidentical nucleotide or amino acid residues at corresponding positionsin two or more sequences when the sequences are aligned to maximizesequence matching, i.e., taking into account gaps and insertions.Identity can be readily calculated by known methods, including but notlimited to those described in: Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing:Informatics and Genome Projects, Smith, D. W., ed., Academic Press, NewYork, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M.,and Griffin, H. G., eds., Humana Press, New Jersey, 1994; SequenceAnalysis in Molecular Biology, von Heinje, G., Academic Press, 1987; andSequence Analysis Primer, Gribskov, M. and Devereux, J., eds., MStockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J.Applied Math., 48: 1073 (1988). Methods to determine identity aredesigned to give the largest match between the sequences tested.Moreover, methods to determine identity are codified in publiclyavailable computer programs.

Optimal alignment of sequences for comparison can be conducted, forexample, by the local homology algorithm of Smith & Waterman, by thehomology alignment algorithms, by the search for similarity method or,by computerized implementations of these algorithms (GAP, BESTFIT,PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys,Inc., San Diego, Calif., United States of America), or by visualinspection. See generally, Altschul, S. F. et al., J. Mol. Biol. 215:403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402(1997).

One example of an algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in (Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894;and Altschul, S. et al., J. Mol. Biol. 215: 403-410 (1990). Software forperforming BLAST analyses is publicly available through the NationalCenter for Biotechnology Information. This algorithm involves firstidentifying high scoring sequence pairs (HSPs) by identifying shortwords of length W in the query sequence, which either match or satisfysome positive-valued threshold score T when aligned with a word of thesame length in a database sequence. T is referred to as the neighborhoodword score threshold.

These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always; 0) and N (penalty scorefor mismatching residues; always; 0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a word length (W) of11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a word length (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide or amino acidsequences would occur by chance. For example, a test nucleic acidsequence is considered similar to a reference sequence if the smallestsum probability in a comparison of the test nucleic acid sequence to thereference nucleic acid sequence is in one embodiment less than about0.1, in another embodiment less than about 0.01, and in still anotherembodiment less than about 0.001.

As used herein, the terms “humanized,” “humanization,” and the like,refer to grafting of the murine monoclonal antibody CDRs disclosedherein to human FRs and constant regions. Also encompassed by theseterms are possible further modifications to the murine CDRs, and humanFRs, by the methods disclosed in, for example, Kashmiri et al. (2005)Methods 36(1):25-34 and Hou et al. (2008) J. Biochem. 144(1):115-120,respectively, to improve various antibody properties, as discussedbelow.

As used herein, the term “humanized antibodies” refers to mAbs andantigen binding fragments thereof, including antibody compounds, thathave binding and functional properties similar to those disclosedherein, and that have FRs and constant regions that are substantiallyhuman or fully human surrounding CDRs derived from a non-human antibody.

As used herein, the term “FR” or “framework sequence” refers to any oneof FRs 1 to 4. Humanized antibodies and antigen binding fragmentsencompassed by the present disclosure include molecules wherein any oneor more of FRs 1 to 4 is substantially or fully human, i.e., wherein anyof the possible combinations of individual substantially or fully humanFRs 1 to 4, is present. For example, this includes molecules in whichFR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially orfully human. Substantially human frameworks are those that have at least80% sequence identity to a known human germline framework sequence.Preferably, the substantially human frameworks have at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequenceidentity, to a framework sequence disclosed herein, or to a known humangermline framework sequence.

Fully human frameworks are those that are identical to a known humangermline framework sequence. Human FR germline sequences can be obtainedfrom the international ImMunoGeneTics (IMGT) database and from TheImmunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc,Academic Press, 2001, the contents of which are herein incorporated byreference in their entirety.

The Immunoglobulin Facts Book is a compendium of the human germlineimmunoglobulin genes that are used to create the human antibodyrepertoire, and includes entries for 203 genes and 459 alleles, with atotal of 837 displayed sequences. The individual entries comprise allthe human immunoglobulin constant genes, and germline variable,diversity, and joining genes that have at least one functional or openreading frame allele, and which are localized in the three major loci.For example, germline light chain FRs can be selected from the groupconsisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27,IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1-5, IGKV1-12, IGKV1D-16,IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39, IGKV1D-33,and IGKJ1-5; and germline heavy chain FRs can be selected from the groupconsisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26,IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3-15, IGHV3-20,IGHV3-66, IGHV3-72, IGHV3-74, IGHV4-31, IGHV3-21, IGHV3-23, IGHV3-30,IGHV3-48, IGHV4-39, IGHV4-59, IGHV5-51, and IGHJ1-6.

Substantially human FRs are those that have at least 80% sequenceidentity to a known human germline FR sequence. Preferably, thesubstantially human frameworks have at least 85%, at least 90%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity, to a framework sequences disclosed herein, or to a known humangermline framework sequence.

CDRs encompassed by the present disclosure include not only thosespecifically disclosed herein, but also CDR sequences having sequenceidentities of at least 80%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto a CDR sequence disclosed herein. Alternatively, CDRs encompassed bythe present disclosure include not only those specifically disclosedherein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acidchanges at corresponding positions compared to CDR sequences disclosedherein. Such sequence identical, or amino acid modified, CDRs preferablybind to the antigen recognized by the intact antibody.

Humanized antibodies in addition to those disclosed herein exhibitingsimilar functional properties according to the present disclosure can begenerated using several different methods, including those disclosed byAlmagro et al. (Frontiers in Biosciences. Humanization of antibodies.(2008) Jan. 1; 13:1619-33).

In one approach, the parent antibody compound CDRs are grafted into ahuman framework that has a high sequence identity with the parentantibody compound framework. The sequence identity of the new frameworkwill generally be at least 80%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95%, at least 96%, at least97%, at least 98%, or at least 99% sequence identical to the sequence ofthe corresponding framework in the parent antibody compound. In the caseof frameworks having fewer than 100 amino acid residues, one, two,three, four, five, six, seven, eight, nine, or ten amino acid residuescan be changed. This grafting may result in a reduction in bindingaffinity compared to that of the parent antibody. If this is the case,the framework can be back-mutated to the parent framework at certainpositions based on specific criteria disclosed by Queen et al. (1991)Proc. Natl. Acad. Sci. USA 88:2869. Additional references describingmethods useful to generate humanized variants based on homology and backmutations include as described in Olimpieri et al. (Bioinformatics 2015Feb. 1; 31(3):434-435) and U.S. Pat. Nos. 4,816,397, 5,225,539, and5,693,761; and the method of Winter and co-workers (Jones et al. (1986)Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; andVerhoeyen et al. (1988) Science 239:1534-1536).

Humanization began with chimerization, a method developed during thefirst half of the 1980's (Morrison, S. L., M. J. Johnson, L. A.Herzenberg & V. T. Oi: Chimeric human antibody molecules: mouseantigen-binding domains with human constant region domains. Proc. Natl.Acad. Sci. USA 81, 6851-5 (1984)), consisting of combining the variable(V) domains of murine antibodies with human constant (C) domains togenerate molecules with ˜70% of human content.

Several different methods can be used to generate humanized antibodies,which are described herein. In one approach, the parent antibodycompound CDRs are grafted into a human FR that has a high sequenceidentity with the parent antibody compound framework. The sequenceidentity of the new FR will generally be at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%identical to the sequence of the corresponding FR in the parent antibodycompound. In the case of FRs having fewer than 100 amino acid residues,one, two, three, four, five, or more amino acid residues can be changed.This grafting may result in a reduction in binding affinity compared tothat of the parent antibody. If this is the case, the FR can beback-mutated to the parent framework at certain positions based onspecific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad.Sci. USA 88:2869. Additional references describing methods useful togenerate humanized variants based on homology and back mutations includeas described in Olimpieri et al. (Bioinformatics. 2015 Feb. 1;31(3):434-435) and U.S. Pat. Nos. 4,816,397, 5,225,539, and 5,693,761;and the method of Winter and co-workers (Jones et al. (1986) Nature321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyenet al. (1988) Science 239:1534-1536).

The identification of residues to consider for back-mutation can becarried out as described below. When an amino acid falls under thefollowing category, the framework amino acid of the human germ-linesequence that is being used (the “acceptor FR”) is replaced by aframework amino acid from a framework of the parent antibody compound(the “donor FR”):

(a) the amino acid in the human FR of the acceptor framework is unusualfor human frameworks at that position, whereas the corresponding aminoacid in the donor immunoglobulin is typical for human frameworks at thatposition;

(b) the position of the amino acid is immediately adjacent to one of theCDRs; or

(c) any side chain atom of a framework amino acid is within about 5-6angstroms (center-to-center) of any atom of a CDR amino acid in a threedimensional immunoglobulin model.

When each of the amino acids in the human FR of the acceptor frameworkand a corresponding amino acid in the donor framework is generallyunusual for human frameworks at that position, such amino acid can bereplaced by an amino acid typical for human frameworks at that position.This back-mutation criterion enables one to recover the activity of theparent antibody compound.

Another approach to generating humanized antibodies exhibiting similarfunctional properties to the antibody compounds disclosed hereininvolves randomly mutating amino acids within the grafted CDRs withoutchanging the framework, and screening the resultant molecules forbinding affinity and other functional properties that are as good as, orbetter than, those of the parent antibody compounds. Single mutationscan also be introduced at each amino acid position within each CDR,followed by assessing the effects of such mutations on binding affinityand other functional properties. Single mutations producing improvedproperties can be combined to assess their effects in combination withone another.

Further, a combination of both of the foregoing approaches is possible.After CDR grafting, one can back-mutate specific FRs in addition tointroducing amino acid changes in the CDRs. This methodology isdescribed in Wu et al. (1999, J. Mol. Biol. 294: 151-162).

Applying the teachings of the present disclosure, a person skilled inthe art can use common techniques, e.g., site-directed mutagenesis, tosubstitute amino acids within the presently disclosed CDR and FRsequences and thereby generate further variable region amino acidsequences derived from the present sequences. Up to all naturallyoccurring amino acids can be introduced at a specific substitution site.The methods disclosed herein can then be used to screen these additionalvariable region amino acid sequences to identify sequences having theindicated in vivo functions. In this way, further sequences suitable forpreparing humanized antibodies and antigen-binding portions thereof inaccordance with the present disclosure can be identified. Preferably,amino acid substitution within the frameworks is restricted to one, two,three, four, or five positions within any one or more of the four lightchain and/or heavy chain FRs disclosed herein. Preferably, amino acidsubstitution within the CDRs is restricted to one, two, three, four, orfive positions within any one or more of the three light chain and/orheavy chain CDRs. Combinations of the various changes within these FRsand CDRs described above are also possible.

That the functional properties of the antibody compounds generated byintroducing the amino acid modifications discussed above conform tothose exhibited by the specific molecules disclosed herein can beconfirmed by the methods in Examples disclosed herein.

As described above, to circumvent the problem of eliciting humananti-murine antibody (HAMA) response in patients, murine antibodies havebeen genetically manipulated to progressively replace their murinecontent with the amino acid residues present in their human counterpartsby grafting their complementarity determining regions (CDRs) onto thevariable light (VL) and variable heavy (VH) frameworks of humanimmunoglobulin molecules, while retaining those murine frameworkresidues deemed essential for the integrity of the antigen-combiningsite. However, the xenogeneic CDRs of the humanized antibodies may evokeanti-idiotypic (anti-Id) response in patients.

To minimize the anti-Id response, a procedure to humanize xenogeneicantibodies by grafting onto the human frameworks only the CDR residuesmost crucial in the antibody-ligand interaction, called “SDR grafting”,has been developed, wherein only the crucial specificity determiningresidues (SDRs) of CDRS are grafted onto the human frameworks.

This procedure, described in Kashmiri et al. (2005, Methods36(1):25-34), involves identification of SDRs through the help of adatabase of the three-dimensional structures of the antigen-antibodycomplexes of known structures, or by mutational analysis of theantibody-combining site. An alternative approach to humanizationinvolving retention of more CDR residues is based on grafting of the‘abbreviated’ CDRs, the stretches of CDR residues that include all theSDRs. Kashmiri et al. also discloses a procedure to assess thereactivity of humanized antibodies to sera from patients who had beenadministered the murine antibody.

Another strategy for constructing human antibody variants with improvedimmunogenic properties is disclosed in Hou et al. (2008, J. Biochem.144(1):115-120). These authors developed a humanized antibody from 4C8,a murine anti-human CD34 monoclonal antibody, by CDR grafting using amolecular model of 4C8 built by computer-assisted homology modelling.Using this molecular model, the authors identified FR residues ofpotential importance in antigen binding. A humanized version of 4C8 wasgenerated by transferring these key murine FR residues onto a humanantibody framework that was selected based on homology to the murineantibody FR, together with the murine CDR residues. The resultinghumanized antibody was shown to possess antigen-binding affinity andspecificity similar to that of the original murine antibody, suggestingthat it might be an alternative to murine anti-CD34 antibodies routinelyused clinically.

Embodiments of the present disclosure encompass antibodies created toavoid recognition by the human immune system containing CDRs disclosedherein in any combinatorial form such that contemplated mAbs can containthe set of CDRs from a single murine mAb disclosed herein, or light andheavy chains containing sets of CDRs comprising individual CDRs derivedfrom two or three of the disclosed murine mAbs. Such mAbs can be createdby standard techniques of molecular biology and screened for desiredactivities using assays described herein. In this way, the disclosureprovides a “mix and match” approach to create novel mAbs comprising amixture of CDRs from the disclosed murine mAbs to achieve new, orimproved, therapeutic activities.

Monoclonal antibodies or antigen-binding fragments thereof encompassedby the present disclosure that “compete” with the molecules disclosedherein are those that bind human CD47 at site(s) that are identical to,or overlapping with, the site(s) at which the present molecules bind.Competing monoclonal antibodies or antigen-binding fragments thereof canbe identified, for example, via an antibody competition assay. Forexample, a sample of purified or partially purified human CD47extracellular domain can be bound to a solid support. Then, an antibodycompound, or antigen binding fragment thereof, of the present disclosureand a monoclonal antibody or antigen-binding fragment thereof suspectedof being able to compete with such disclosure antibody compound areadded. One of the two molecules is labelled. If the labelled compoundand the unlabelled compound bind to separate and discrete sites on CD47,the labelled compound will bind to the same level whether or not thesuspected competing compound is present. However, if the sites ofinteraction are identical or overlapping, the unlabelled compound willcompete, and the amount of labelled compound bound to the antigen willbe lowered. If the unlabelled compound is present in excess, verylittle, if any, labelled compound will bind. For purposes of the presentdisclosure, competing monoclonal antibodies or antigen-binding fragmentsthereof are those that decrease the binding of the present antibodycompounds to CD47 by about 50%, about 60%, about 70%, about 80%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, or about 99%. Details of procedures for carrying out suchcompetition assays are well known in the art and can be found, forexample, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. Such assays canbe made quantitative by using purified antibodies. A standard curve isestablished by titrating one antibody against itself, i.e., the sameantibody is used for both the label and the competitor. The capacity ofan unlabelled competing monoclonal antibody or antigen-binding fragmentthereof to inhibit the binding of the labelled molecule to the plate istitrated. The results are plotted, and the concentrations necessary toachieve the desired degree of binding inhibition are compared.

Whether mAbs or antigen-binding fragments thereof that compete withantibody compounds of the present disclosure in such competition assayspossess the same or similar functional properties of the presentantibody compounds can be determined via these methods in conjunctionwith the methods described in Examples below. In various embodiments,competing antibodies for use in the therapeutic methods encompassedherein possess biological activities as described herein in the range offrom about 50% to about 100% or about 125%, or more, compared to that ofthe antibody compounds disclosed herein. In some embodiments, competingantibodies possess about 50%, about 60%, about 70%, about 80%, about85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, about 99%, or identical biologicalactivity compared to that of the antibody compounds disclosed herein asdetermined by the methods disclosed in the Examples presented below.

The mAbs or antigen-binding fragments thereof, or competing antibodiesuseful in the compositions and methods can be any of the isotypesdescribed herein. Furthermore, any of these isotypes can comprisefurther amino acid modifications as follows.

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG1 isotype.

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to alter antibody half-life. Antibody half-life isregulated in large part by Fc-dependent interactions with the neonatalFc receptor (Roopenian and Alikesh, 2007). The human IgG1 constantregion of the monoclonal antibody, antigen-binding fragment thereof, orcompeting antibody can be modified to increase half-life include, butare not limited to amino acid modifications N434A, T307A/E380A/N434A(Petkova et al., 2006, Yeung et al., 2009); M252Y/S254T/T256E(Dall'Acqua et al., 2006); T250Q/M428L (Hinton et al., 2006); andM428L/N434S (Zalevsky et al., 2010).

As opposed to increasing half-life, there are some circumstances wheredecreased half-life would be desired, such as to reduce the possibilityof adverse events associated with high Antibody-Dependent CellularCytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC)antibodies (Presta 2008). The human IgG1 constant region of themonoclonal antibody, antigen-binding fragment thereof, or competingantibody described herein can be modified to decrease half-life and/ordecrease endogenous IgG include, but are not limited to amino acidmodifications I253A (Petkova et al., 2006); P257I/N434H, D376V/N434H(Datta-Mannan et al., 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaroet al., 2005).

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase or decrease antibody effector functions.These antibody effector functions include, but are not limited to,Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-DependentCytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), C1qbinding, and altered binding to Fc receptors.

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase antibody effector function include, but arenot limited to amino acid modifications S298A/E333A/K334 (Shields etal., 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al., 2006);F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, andF243L/R292P/Y300L/V305I/P396L (Stevenhagen et al., 2007); G236A,G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al., 2008);K326A/E333A, K326A/E333S and K326W/E333S (Idusogie et al., 2001); S267Eand S267E/L328F (Smith et al., 2012); H268F/S324T, S267E/H268F,S267E/S234T, and S267E/H268F/S324T (Moore et al., 2010); S298G/T299A(Sazinsky et al., 2008); E382V/M428I (Jung et al., 2010).

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector function include, but arenot limited to amino acid modifications N297A and N297Q (Bolt et al.,1993, Walker et al., 1989); L234A/L235A (Xu et al., 2000);K214T/E233P/L234V/L235A/G236-deleted/A327G/P331A/D356E/L358M (Ghevaertet al., 2008); C226S/C229S/E233P/L234V/L235A (McEarchern et al., 2007);S267E/L328F (Chu et al., 2008).

The human IgG1 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector function include, but arenot limited to amino acid modifications V234A/G237A (Cole et al., 1999);E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331S,H268Q/A330S/V309L/P331S, H268D/A330S/V309L/P331S,H268Q/A330R/V309L/P331S, H268D/A330R/V309L/P331S, E233D/A330R,E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G237D/H268D/P271G,G237D/H268Q/P271G, G237D/P271G/A330R, G237D/P271G/A330S,E233D/H268D/P271G/A330R, E233D/H268Q/P271G/A330R,E233D/H268D/P271G/A330S, E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R, G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S,G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R,E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S,E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R, P238D/E233D/A330S,P238D/E233D/P271G/A330R, P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R,P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R,P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S,P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R,P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S,P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R,P238D/E233D/G237D/H268Q/P271 G/A330R,P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S(An et al., 2009, Mimoto, 2013).

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG2 isotype.

The human IgG2 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase or decrease antibody effector functions.These antibody effector functions include, but are not limited to,Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-DependentCytotoxicity (CDC), Antibody-Dependent Cellular Phagocytosis (ADCP), andC1q binding, and altered binding to Fc receptors.

The human IgG2 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to increase antibody effector function include, but arenot limited to the amino acid modification K326A/E333S (Idusogie et al.,2001).

The human IgG2 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector function include, but arenot limited to amino acid modifications V234A/G237A (Cole et al., 1999);V234A, G237A, P238S, H268A, E233D, G237D, P238D, H268Q, H268D, P271G,V309L, A330S, A330R, P331S, P238S/H268A,V234A/G237A/P238S/H268A/V309L/A330S/P331S, H268Q/A330S/V309L/P331S,H268D/A330S/V309L/P331S, H268Q/A330R/V309L/P331S,H268D/A330R/V309L/P331S, E233D/A330R, E233D/A330S, E233D/P271G/A330R,E233D/P271G/A330S, G237D/H268D/P271G, G237D/H268Q/P271G,G237D/P271G/A330R, G237D/P271G/A330S, E233D/H268D/P271G/A330R,E233D/H268Q/P271G/A330R, E233D/H268D/P271G/A330S,E233D/H268Q/P271G/A330S, G237D/H268D/P271G/A330R,G237D/H268Q/P271G/A330R, G237D/H268D/P271G/A330S,G237D/H268Q/P271G/A330S, E233D/G237D/H268D/P271G/A330R,E233D/G237D/H268Q/P271G/A330R, E233D/G237D/H268D/P271G/A330S,E233D/G237D/H268Q/P271G/A330S, P238D/E233D/A330R, P238D/E233D/A330S,P238D/E233D/P271G/A330R, P238D/E233D/P271G/A330S, P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/P271G/A330R,P238D/G237D/P271G/A330S, P238D/E233D/H268D/P271G/A330R,P238D/E233D/H268Q/P271G/A330R, P238D/E233D/H268D/P271G/A330S,P238D/E233D/H268Q/P271G/A330S, P238D/G237D/H268D/P271G/A330R,P238D/G237D/H268Q/P271G/A330R, P238D/G237D/H268D/P271G/A330S,P238D/G237D/H268Q/P271G/A330S, P238D/E233D/G237D/H268D/P271G/A330R,P238D/E233D/G237D/H268Q/P271 G/A330R,P238D/E233D/G237D/H268D/P271G/A330S, P238D/E233D/G237D/H268Q/P271G/A330S(An et al., 2009, Mimoto, 2013).

The Fc region of a human IgG2 of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to alter isoform and/or agonistic activity, include, butare not limited to amino acid modifications C127S (CH1 domain), C232S,C233S, C232S/C233S, C236S, and C239S (White et al., 2015, Lightle etal., 2010).

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG3 isotype.

The human IgG3 constant region of the monoclonal antibody, or antigenbinding fragment thereof, wherein said human IgG3 constant region of themonoclonal antibody, or antigen-binding fragment thereof can be modifiedat one or more amino acid(s) to increase antibody half-life,Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-DependentCytotoxicity (CDC), or apoptosis activity.

The human IgG3 constant region of the monoclonal antibody, orantigen-binding fragment thereof, wherein said human IgG3 constantregion of the monoclonal antibody, or antigen-binding fragment thereofcan be modified at amino acid R435H to increase antibody half-life.

The monoclonal antibody or antigen-binding fragment thereof, orcompeting antibody described herein can be of the human IgG4 isotype.

The human IgG4 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to decrease antibody effector functions. These antibodyeffector functions include, but are not limited to, Antibody-DependentCellular Cytotoxicity (ADCC) and Antibody-Dependent CellularPhagocytosis (ADCP).

The human IgG4 constant region of the monoclonal antibody,antigen-binding fragment thereof, or competing antibody described hereincan be modified to prevent Fab arm exchange and/or decrease antibodyeffector function include, but are not limited to amino acidmodifications F234A/L235A (Alegre et al., 1994); S228P, L235E andS228P/L235E (Reddy et al., 2000).

As used herein, the term “tumor” refers to all neoplastic cell growthand proliferation, whether malignant or benign, and all pre-cancerousand cancerous cells and tissues.

The terms “cancer,” “cancerous,” and “tumor” are not mutually exclusiveas used herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byaberrant cell growth/proliferation. Examples of cancers include, but arenot limited to, carcinoma, lymphoma (i.e., Hodgkin's and non-Hodgkin'slymphoma), blastoma, sarcoma, and leukemia. More particular examples ofsuch cancers include squamous cell cancer, small-cell lung cancer,non-small cell lung cancer, adenocarcinoma of the lung, squamouscarcinoma of the lung, cancer of the peritoneum, hepatocellular cancer,gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer,colon cancer, colorectal cancer, endometrial or uterine carcinoma,salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer,vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and otherlymphoproliferative disorders, and various types of head and neckcancer.

The term “susceptible cancer” as used herein refers to a cancer, cellsof which express CD47, and are responsive to treatment with an anti-CD47antibody or antigen binding fragment thereof, or competing antibody orantigen binding fragment thereof, of the present disclosure.

“Nitric oxide (NO) donor, precursor, or nitric oxide generating topicalagent” refers to a compound or agent that either delivers NO, or thatcan be converted to NO through enzymatic or non-enzymatic processes.Examples include, but are not limited to, NO gas, isosorbide dinitrite,nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1),S-nitroso-N-acetyl-penicillamine (SNAP), Diethylenetriamine/NO(DETA/NO), S-nitrosothiols, Bidil®, and arginine.

“Soluble guanylyl cyclase (sGC)” is the receptor for nitric oxide invascular smooth muscle. In the cardiovascular system, nitric oxide isendogenously generated by endothelial nitric oxide synthase fromL-arginine, and activates soluble guanylyl cyclase in adjacent vascularsmooth muscle cells to increase cGMP levels, inducing vascularrelaxation. Nitric oxide binds to the normally reduced heme moiety ofsoluble guanylyl cyclase, and increases the formation of cGMP from GTP,leading to a decrease in intracellular calcium, vasodilation, andanti-inflammatory effects. Oxidation of the heme iron on sGC decreasesresponsiveness of the enzyme to nitric oxide, and promotesvasoconstriction. The nitric oxide-sGC-cGMP pathway therefore plays animportant role in cardiovascular diseases. Nitrogen-containing compoundssuch as sodium azide, sodium nitrite, hydroxylamine, nitroglycerin, andsodium nitroprusside have been shown to stimulate sGC, causing anincrease in cGMP, and vascular relaxation. In contrast to stimulators ofsGC, which bind to reduced sGC, activators of sGC activate the oxidizedor heme-deficient sGC enzyme that is not responsive to nitric oxide,i.e., they stimulate sGC independent of redox state. While stimulatorsof of sGC can enhance the sensitivity of reduced sGC to nitric oxide,activators of sGC can increase sGC enzyme activity even when the enzymeis oxidized and is therefore less, or unresponsive, to nitric oxide.Thus, sGC activators are non-nitric oxide based. Note the reviews ofNossaman et al. (2012) Critical Care Research and Practice, Volume 2012,article 290805, and Derbyshire and Marletta (2012) Ann. Rev. Biochem.81:533-559.

“An agent that activates soluble guanylyl cyclase” refers, for example,to organic nitrates (Artz et al. (2002) J. Biol. Chem. 277:18253-18256);protoporphyrin IX (Ignarro et al. (1982) Proc. Natl. Acad. Sci. USA79:2870-2873); YC-1 (Ko et al. (1994) Blood 84:4226-4233); BAY 41-2272and BAY 41-8543 (Stasch et al. (2001 Nature 410 (6825): 212-5),CMF-1571, and A-350619 (reviewed in Evgenov et al. (2006) Nat. Rev.Drug. Discov. 5:755-768); BAY 58-2667 (Cinaciguat; Frey et al. (2008)Journal of Clinical Pharmacology 48 (12): 1400-10); BAY 63-2521(Riociguat; Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65).Additional soluble guanylyl cyclase activators are disclosed in Staschet al. (2011) Circulation 123:2263-2273; Derbyshire and Marletta (2012)Ann. Rev. Biochem. 81:533-559, and Nossaman et al. (2012) Critical CareResearch and Practice, Volume 2012, Article ID 290805, pages 1-12.

cGMP can also be increased by inhibiting degradation usingphosphodiesterase inhibitors. Examples of “an agent that inhibits cyclicnucleotide phosphodiesterases” include, tadalafil, vardenafil, udenafil,and sildenafil avanafil.

As used herein, term “treating” or “treat” or “treatment” means slowing,interrupting, arresting, controlling, stopping, reducing, or reversingthe progression or severity of a sign, symptom, disorder, condition, ordisease, but does not necessarily involve a total elimination of alldisease-related signs, symptoms, conditions, or disorders. The term“treating” and the like refer to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop.

As used herein, term “effective amount” refers to the amount or dose ofan antibody compound of the present disclosure which, upon single ormultiple dose administration to a patient or organ, provides the desiredtreatment or prevention.

The precise effective amount for any particular subject will depend upontheir size and health, the nature and extent of their condition, and thetherapeutics or combination of therapeutics selected for administration.The effective amount for a given patient is determined by routineexperimentation and is within the judgment of a clinician.Therapeutically effective amounts of the present antibody compounds canalso comprise an amount in the range of from about 0.1 mg/kg to about150 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kgto about 50 mg/kg, or from about 0.05 mg/kg to about 10 mg/kg per singledose administered to a harvested organ or to a patient. Knownantibody-based pharmaceuticals provide guidance in this respect. Forexample, Herceptin™ is administered by intravenous infusion of a 21mg/ml solution, with an initial loading dose of 4 mg/kg body weight anda weekly maintenance dose of 2 mg/kg body weight; Rituxan™ isadministered weekly at 375 mg/m2; for example.

A therapeutically effective amount for any individual patient can bedetermined by the health care provider by monitoring the effect of theantibody compounds on tumor regression, circulating tumor cells, tumorstem cells or anti-tumor responses. Analysis of the data obtained bythese methods permits modification of the treatment regimen duringtherapy so that optimal amounts of antibody compounds of the presentdisclosure, whether employed alone or in combination with one another,or in combination with another therapeutic agent, or both, areadministered, and so that the duration of treatment can be determined aswell. In this way, the dosing/treatment regimen can be modified over thecourse of therapy so that the lowest amounts of antibody compounds usedalone or in combination that exhibit satisfactory efficacy areadministered, and so that administration of such compounds is continuedonly so long as is necessary to successfully treat the patient. Knownantibody-based pharmaceuticals provide guidance relating to frequency ofadministration e.g., whether a pharmaceutical should be delivered daily,weekly, monthly, etc. Frequency and dosage may also depend on theseverity of symptoms.

In some embodiments antibody compounds of the present disclosure can beused as medicaments in human and veterinary medicine, administered by avariety of routes including, but not limited to, oral, intravenous,intramuscular, intra-arterial, intramedullary, intraperitoneal,intrathecal, intraventricular, transdermal, transcutaneous, topical,subcutaneous, intratumoral, intranasal, enteral, sublingual,intravaginal, intravesiciular or rectal routes. The compositions canalso be administered directly into a lesion such as a tumor. Dosagetreatment may be a single dose schedule or a multiple dose schedule.Hypo sprays may also be used to administer the pharmaceuticalcompositions. Typically, the therapeutic compositions can be prepared asinjectables, either as liquid solutions or suspensions. Solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection can also be prepared. Veterinary applications include thetreatment of companion/pet animals, such as cats and dogs; workinganimals, such as guide or service dogs, and horses; sport animals, suchas horses and dogs; zoo animals, such as primates, cats such as lionsand tigers, bears, etc.; and other valuable animals kept in captivity.

Such pharmaceutical compositions can be prepared by methods well knownin the art. See, e.g., Remington: The Science and Practice of Pharmacy,21st Edition (2005), Lippincott Williams & Wilkins, Philadelphia, Pa.,and comprise one or more antibody compounds disclosed herein, and apharmaceutically or veterinarily acceptable, for example,physiologically acceptable, carrier, diluent, or excipient.

The present disclosure describes anti-CD47 mAbs with distinct functionalprofiles. These antibodies possess distinct combinations of propertiesselected from the following: These antibodies possess distinctcombinations of properties selected from the following: 1) exhibitcross-reactivity with one or more species homologs of CD47; 2) block theinteraction between CD47 and its ligand SIRPα; 3) increase phagocytosisof human tumor cells; 4) induce death of susceptible human tumor cells;5) do not induce cell death of human tumor cells; 6) do not have reducedor minimal binding to human red blood cells (hRBCs); 7) have reducedbinding to hRBCs; 8) have minimal binding to hRBCs; 9) cause reducedagglutination of hRBCs; 10) cause no detectable agglutination of hRBCs;11) reverse TSP1 inhibition of the nitric oxide (NO) pathway; 12) do notreverse TSP1 inhibition of the NO pathway; 13) cause loss ofmitochondrial membrane potential; 14) do not cause cause loss ofmitochondrial membrane potential; 15) cause an increase in cell surfacecalreticulin expression on human tumor cells; 16) do not cause anincrease in cell surface calreticulin expression on human tumor cells;17) cause an increase in adenosine triphosphate (ATP) release by humantumor cells; 18) do not cause an increase in adenosine triphosphate(ATP) release by human tumor cells; 19) cause an increase in highmobility group box 1 (HMGB1) release by human tumor cells; 20) do notcause an increase in high mobility group box 1 (HMGB1) release by humantumor cells; 21) cause an increase in type I interferon release by humantumor cells; 22) do not cause an increase in type I interferon releaseby human tumor cells; 23) cause an increase in C-X-C Motif ChemokineLigand 10 (CXCL10) release by human tumor cells; 24) do not cause anincrease in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by humantumor cells; 25) cause an increase in cell surface proteindisulfide-isomerase A3 (PDIA3) expression on human tumor cells; 26) donot cause an increase in cell surface protein disulfide-isomerase A3(PDIA3) expression on human tumor cells; 27) cause an increase in cellsurface heat shock protein 70 (HSP70) expression on human tumor cells;28) do not cause an increase in cell surface heat shock protein 70(HSP70) expression on human tumor cells; 29) cause an increase in cellsurface heat shock protein 90 (HSP90) expression on human tumor cells;30) do not cause an increase in cell surface heat shock protein 90(HSP90) expression on human tumor cells; 31) have reduced binding tonormal human cells, which includes, but is not limited to, endothelialcells, skeletal muscle cells, epithelial cells, and peripheral bloodmononuclear cells (e.g., human aortic endothelial cells, human skeletalmuscle cells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, and humanperipheral blood mononuclear cells); 32) do not have reduced binding tonormal human cells, which includes, but is not limited to, endothelialcells, skeletal muscle cells, epithelial cells, and peripheral bloodmononuclear cells (e.g., human aortic endothelial cells, human skeletalmuscle cells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, and humanperipheral blood mononuclear cells); 33) have a greater affinity forhuman CD47 at an acidic pH compared to physiological pH; 34) do not havea greater affinity for human CD47 at an acidic pH compared tophysiological pH; and 35) cause an increase in annexin A1 release byhuman tumor cells.

The anti-CD47 antibodies and antigen binding fragments thereof of thepresent disclosure possess combinations of properties that are distinctfrom the anti-CD47 antibodies of the prior art. These properties andcharacteristics will now be described in further detail. As used herein,the term “binds to human CD47” refers to binding with an apparent Kdgreater than 50 nM, for example, in a solid phase ELISA assay or cellbased assay.

As used herein, the terms “apparent binding affinity and apparent Kd”are determined by non-linear fit (Prism GraphPad software) of thebinding data at the various antibody concentrations.

Binding to CD47 of Different Species

The anti-CD47 antibodies, and antigen binding fragments thereof, of thepresent disclosure bind human CD47. In certain embodiments, theanti-CD47 antibodies exhibit cross-reactivity with one or more specieshomologs of CD47, for example CD47 homologs of non-human primate origin.In certain embodiments, the anti-CD47 antibodies and antigen bindingfragments thereof of the present disclosure bind to human CD47 and toCD47 of non-human primate, mouse, rat, and/or rabbit origin. Thecross-reactivity with other species homologs can be particularlyadvantageous in the development and testing of therapeutic antibodies.For example, pre-clinical toxicology testing of therapeutic antibodiesis frequently carried out in non-human primate species including, butnot limited to, cynomolgus monkey, green monkey, rhesus monkey andsquirrel monkey. Cross-reactivity with these species homologs cantherefore be particularly advantageous for the development of antibodiesas clinical candidates.

As used herein, the term “cross-reacts with one or more species homologsof CD47” refers to binding with an apparent Kd greater than 50 nM.

Blocking the Interaction Between CD47 and SIRPα and PromotingPhagocytosis

CD47, also known as integrin associated protein (IAP), is a 50 kDa cellsurface receptor that is comprised of an extracellular N-terminal IgVdomain, a five membrane-spanning transmembrane domain, and a shortC-terminal intracellular tail that is alternatively spliced.

Two ligands bind to CD47: Signal Regulatory Protein alpha (SIRPα) andThrombospondin-1 (TSP1). TSP1 is present in plasma and synthesized bymany cells, including platelets. SIRPα is expressed on hematopoieticcells, which include macrophages and dendritic cells.

When SIRPα on a phagocyte engages CD47 on a target cell, thisinteraction prevents phagocytosis of the target cell. The interaction ofCD47 and SIRPα effectively sends a “don't eat me” signal to thephagocyte (Oldenborg et al. Science 288: 2051-2054, 2000). Blocking theinteraction of SIRPα and CD47 with an anti-CD47 mAb in a therapeuticcontext can provide an effective anti-cancer treatment by promoting theuptake and clearance of cancer cells by the host's immune system. Thus,an important functional characteristic of some anti-CD47 mAbs is theability to block the interaction of CD47 and SIRPα, resulting inphagocytosis of CD47 expressing tumor cells by phagocytes includingmacrophages. Several anti-CD47 mAbs have been shown to block theinteraction of CD47 and SIRPα, including B6H12 (Seiffert et al. Blood94:3633-3643,1999; Latour et al. J. Immunol. 167: 2547-2554, 2001;Subramanian et al. Blood 107: 2548-2556, 2006; Liu et al. J Biol. Chem.277: 10028-10036, 2002; Rebres et al. J. Cellular Physiol. 205: 182-193,2005), BRIC126 (Vernon-Wilson et al. Eur J Immunol. 30: 2130-2137, 2000;Subramanian et al. Blood 107: 2548-2556, 2006), CC2C6 (Seiffert et al.Blood 94:3633-3643,1999), 1F7 (Rebres et al. J. Cellular Physiol. 205:182-193, 2005), 5F9 (Liu et al. PLoS One. 2015 Sep. 21; 10(9): e0137345)and CC-90002 (Narla et al. Proc Am Assoc Cancer Res 58: 1200, 2017; abst469)4. B6H12 and BRIC126 have also been shown to cause phagocytosis ofhuman tumor cells by human and mouse macrophages (Willingham et al. ProcNatl Acad Sci USA 109(17):6662-6667, 2012; Chao et al. Cell 142:699-713,2012; EP 2 242 512 B1). Other existing anti-CD47 mAbs, such as 2D3, doesnot block the interaction of CD47 and SIRPα (Seiffert et al. Blood94:3633-3643,1999; Latour et al. J. Immunol. 167: 2547-2554, 2001;Rebres et al. J. Cellular Physiol. 205: 182-193, 2005), and does notcause phagocytosis of tumor cells (Willingham et al. Proc Natl Acad SciUSA 109(17):6662-6667, 2012; Chao et al. Cell 142:699-713, 2012; EP 2242 512 B1).

As used herein, the term “blocks SIRPα binding to human CD47” refers toa greater than 50% reduction of SIRPα-Fc binding to CD47 on cells by ananti-CD47 mAb compared to either untreated cells or cells treated with anegative antibody.

The anti-CD47 mAbs of the disclosure described herein, block theinteraction of CD47 and SIRPα and increase phagocytosis of human tumorcells.

“Phagocytosis” of cancer cells refers to the engulfment and digestion ofsuch cells by phagocytes including, but not limited to, macrophages anddendritic cells, and the eventual digestion or degradation of thesecancer cells and the release of digested or degraded cellular componentsextracellularly, or intracellularly to undergo further processing.Anti-CD47 monoclonal antibodies that block SIRPα binding to CD47increase phagocytosis of cancer cells. SIRPα binding to CD47 on cancercells would otherwise allow these cells to escape phagocytosis. Thecancer cell may be viable or living cancer cells.

As used herein, the term “increases phagocytosis of human tumor cells”refers to a greater than 2-fold increase in phagocytosis of human tumorcells by human macrophages in the presence of an anti-CD47 mAb comparedto either untreated cells or cells treated with a negative controlantibody.

Inducing Death of Tumor Cells

Some soluble anti-CD47 mAbs initiate a cell death program on binding toCD47 on tumor cells, resulting in collapse of mitochrondrial membranepotential, loss of ATP generating capacity, increased cell surfaceexpression of phosphatidylserine (detected by increased staining forannexin V) and cell death without the participation of caspases orfragmentation of DNA. Such soluble anti-CD47 mAbs have the potential totreat a variety of solid and hematological cancers. Several solubleanti-CD47 mAbs which have been shown to induce tumor cell death,including MABL-1, MABL-2 and fragments thereof (U.S. Pat. No. 8,101,719;Uno et al. Oncol Rep. 17: 1189-94, 2007; Kikuchi et al. Biochem BiophysRes. Commun. 315: 912-8, 2004), Ad22 (Pettersen et al. J. Immuno. 166:4931-4942, 2001; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003), and1F7 (Manna et al. J. Immunol. 170: 3544-3553, 2003; Manna et al. CancerResearch, 64: 1026-1036, 2004). Some of the anti-CD47 mAbs of thedisclosure described herein induce cell death of human tumor cells.

Induction of cell death refers to the ability of certain of the solubleanti-CD47 antibodies, murine antibodies, chimeric antibodies, humanizedantibodies, or antigen-binding fragments thereof (and competingantibodies and antigen-binding fragments thereof) disclosed herein tokill cancer cells via a cell autonomous mechanism without participationof complement or other cells including, but not limited to, T cells,neutrophils, natural killer cells, macrophages, or dendritic cells.

The terms “inducing cell death” or “kills” and the like, are usedinterchangeably herein.

As used herein, the term “induces death of human tumor cells” refers toincreased binding of annexin V (in the presence of calcium) andincreased 7-aminoactinomycin D (7-AAD) or propidium iodide uptake inresponse to treatment with an anti-CD47 mAb. These features may bequantitated in three cell populations: annexin V positive (annexin V⁺),annexin V positive/7-AAD negative (annexin V⁺/7-AAD⁻) and annexin Vpositive/7-AAD positive (annexin V⁺/7-AAD⁺) by flow cytometry. Inductionof cell death may be defined by a greater than 2-fold increase in eachof the above cell populations in human tumor cells caused by solubleanti-CD47 mAb compared to the background obtained with the negativecontrol antibody, (humanized, isotype-matched antibody) or untreatedcells.

Another indicator of cell death is loss of mitochondrial function andmembrane potential by the tumor cells as assayed by one of severalavailable measures (potentiometric fluorescent dyes such as DiO-C6 orJC1 or formazan-based assays such as MTT or WST-1).

As used herein, the term “causes loss of mitochondrial membranepotential” refers to a statistically significant (p<0.05) decrease inmitochondrial membrane potential by a soluble anti-CD47 mAb compared tothe background obtained with a negative control, humanizedisotype-matched antibody or no treatment.

Among the present humanized or chimeric mAbs, those that induce celldeath of human tumor cells cause increased annexin V binding similar tothe findings reported for anti-CD47 mAbs Ad22 (Pettersen et al. J.Immunol. 166: 4931-4942, 2001; Lamy et al. J. Biol. Chem. 278:23915-23921, 2003); 1F7 (Manna and Frazier J. Immunol. 170:3544-3553,2003; Manna and Frazier Cancer Res. 64:1026-1036, 2004); and MABL-1 and2 (U.S. Pat. No. 7,531,643 B2; U.S. Pat. No. 7,696,325 B2; U.S. Pat. No.8,101,719 B2).

Cell viability assays are described in NCI/NIH guidance manual thatdescribes numerous types of cell based assays that can be used to assessinduction of cell death caused by CD47 antibodies: “Cell ViabilityAssays”, Terry L Riss, PhD, Richard A Moravec, BS, Andrew L Niles, MS,Helene A Benink, PhD, Tracy J Worzella, MS, and Lisa Minor, PhD.Contributor Information, published May 1, 2013.

Binding to hRBCs

CD47 is expressed on human erythrocytes (hRBCs) (Brown. J Cell Biol.111: 2785-2794, 1990; Avent. Biochem J., (1988) 251: 499-505; Knapp.Blood, (1989) Vol. 74, No. 4, 1448-1450; Oliveira et al. Biochimica etBiophysica Acta 1818: 481-490, 2012; Petrova P. et al. Cancer Res 2015;75(15 Suppl): Abstract no. 4271). It has been shown that anti-CD47 mAbsbind to RBCs, including B6H12 (Brown et al. J. Cell Biol., 1990,Oliveira et al. Biochimica et Biophysica Acta 1818: 481-490, 2012,Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract no. 4271),BRIC125 (Avent. Biochem J., (1988) 251: 499-505), BRIC126 (Avent.Biochem J., (1988) 251: 499-505; Petrova P. et al. Cancer Res 2015;75(15 Suppl): Abstract no. 4271), 5F9 (Uger R. et al. Cancer Res 2014;74(19 Suppl): Abstract no. 5011, Liu et al. PLoS One. 2015 Sep. 21;10(9): e0137345; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract3019)), anti-CD47 antibodies disclosed in US Patent Publication2014/0161799, WO Publication 2014/093678, US Patent Publication2014/0363442, and CC2C6 (Petrova P. et al. Cancer Res 2015; 75(15Suppl): Abstract no. 4271, Uger R. et al. Cancer Res 2014; 74(19 Suppl):Abstract no. 5011). It has also been shown that a SIRPα-Fc fusionprotein, which binds to human CD47, has reduced binding to human RBCscompared to other human cells (Uger R. et al. Cancer Res 2014; 74(19Suppl: Abstract no. 5011; Petrova et al. Clin Cancer Res 23: 1068-1079,2017). Binding to RBCs can be reduced by generation of bi-specificantibodies with only one CD47 binding arm (Masternak et al. Cancer Res2015; 75(15 Suppl): Abstract no. 2482). Because some anti-CD47 mAbs havebeen shown to result in reduction of RBCs when administered tocynomolgus monkeys (Mounho-Zamora B. et al. The Toxicologist, Supplementto Toxicological Sciences, 2015; 144 (1): Abstract 596: 127, Liu et al.PLoS One. 2015 Sep. 21; 10(9): e0137345; Pietsch et al. Cancer Res 2015;75(15 Suppl): Abstract nr 2470), it is highly desirable to identifyanti-CD47 mAbs that have reduced or minimal binding to CD47-expressingRBCs.

As used herein, the terms “red blood cell(s)” and “erythrocyte(s)” aresynonymous and used interchangeably herein.

As used herein, the term “reduced binding to hRBCs” refers to anapparent Kd of an anti-CD47 mAb binding to a hRBC which is 8-fold orgreater than the apparent Kd on a human tumor cell, wherein the tumorcell is an OV10 hCD47 cell (human OV10 ovarian cancer cell lineexpressing human CD47).

As used herein, the term “minimal binding” or “MB” refers to nomeasurable binding to hRBCs at an anti-CD47 mAb concentration up to5,000 pM.

Prior to the disclosure described herein, no monospecific anti-CD47 mAbshave been reported that have minimal binding to human RBCs expressingCD47.

Some of the anti-CD47 mAbs, disclosed herein, have reduced or minimalbinding to human RBCs.

Binding to Human Endothelial Cells and Other Normal Human Cells

In addition to expression/overexpression on most hematologicalmalignancies and solid tumors (Willingham et al., Proc Natl Acad Sci2012), CD47 is also expressed, by many but not all, normal cell types,including, but not limited to RBCs (see previous section), lymphocytesand mononuclear cells, endothelial cells, and brain, liver, muscle cellsand/or tissues (Brown et al., J Cell Biol 1990; Reinhold et al., J CellSci. 1995; Matozaki et al., Cell 2009; Stefanidakis et al., Blood 2008;Xiao et al., Cancer Letters 2015). Because of this expression, it isexpected that some anti-CD47 mAbs would bind to these normal celltypes/tissues in addition to the cancer cells which are the therapeutictarget. It is therefore desirable to identify anti-CD47 mAbs that eitherhave reduced or minimal binding to some of these normal cells to bothreduce potential non-desired effects on these normal cells and alsoallow more available antibody for binding to the tumor cells. Anti-CD47mAbs with such reduced or minimal binding to normal cells have not beendescribed.

As used herein, the terms “reduced binding to normal human cells whichincludes, but is not limited to, endothelial cells, skeletal musclecells, epithelial cells, and peripheral blood mononuclear cells (e.g.,human aortic endothelial cells, human skeletal muscle cells, humanmicrovascular endothelial cells, human renal tubular epithelial cells,human peripherial blood CD3+ cells, and human peripheral bloodmononuclear cells) refers to the apparent Kd of an anti-CD47 mAb bindingto these cells which is 8-fold or greater than the apparent Kd of theanti-CD47 mAb binding to a human tumor cell, wherein the tumor cell isOV10 hCD47.

As used herein, the term “minimal binding” or “MB” refers to nomeasurable binding of an antibody or other molecule as described hereinto normal human cells which includes, but is not limited to, endothelialcells, skeletal muscle cells, epithelial cells, and peripheral bloodmononuclear cells (e.g., human aortic endothelial cells, human skeletalmuscle cells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, and humanperipheral blood mononuclear cells) at an anti-CD47 mAb concentration upto 5,000 pM.

Agglutination of RBCs

Red blood cell (RBC) agglutination or hemagglutination is a homotypicinteraction that occurs when RBCs aggregate or clump together followingincubation with various agents, including antibodies to RBC antigens andcell surface proteins such as CD47. Many anti-CD47 antibodies have beenreported to cause hemagglutination of washed human RBCs in vitro, in aconcentration dependent manner, including B6H12, BRIC126, MABL-1,MABL-2, CC2C6, and 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl):Abstract no. 5011, U.S. Pat. No. 9,045,541, Uno et al. Oncol Rep. 17:1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8,2004; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract 3019)).This functional effect requires binding to RBCs by an intact, bivalentantibody and can be reduced or eliminated by generating antibodyfragments, either a F(ab′) or svFv (Uno et al. Oncol Rep. 17: 1189-94,2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8, 2004) orbi-specific antibodies with only one CD47 binding arm (Masternak et al.Cancer Res 2015; 75(15 Suppl): Abstract no. 2482). Other functionalproperties of these fragments, including cell killing, were shown to beeither reduced or retained in these fragments (Uno et al. Oncol Rep. 17:1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8,2004). The mouse antibody 2D3 is an example of an anti-CD47 antibodythat binds to CD47 on red blood cells but does not causehemagglutination (U.S. Pat. No. 9,045,541, Petrova et al. Cancer Res2015; 75(15 Suppl): Abstract no. 4271).

Hemagglutination has been shown to be reduced/eliminated by reducing thebinding selectively to human RBCs, but not other cells, using a SIRPα-Fcfusion protein (Uger R. et al. Blood 2013; 122(21): 3935). In addition,mouse anti-CD47 mAb 2A1 and humanized versions of 2A1 have been reportedto block CD47/SIRPα but do not exhibit hemagglutination activity in awashed RBC assay (U.S. Pat. No. 9,045,541; Narla et al. Proc Am AssocCancer Res 58: 1200, 2017; abst 4694). A small number of a panel ofmouse anti-human CD47 antibodies (8 of 23) were reported to not causehemagglutination of human RBCs (Pietsch E et al. Blood Cancer Journal(2017) 7, e536; doi:10.1038/bcj.2017.7). Therefore, prior to thedisclosure described herein, there was a need to identify CD47 mAbs thatblock SIRPα/CD47 binding, have reduced or minimal binding to RBCs and/orcause no detectable hemagglutination. The term “agglutination” refers tocellular clumping, while the term “hemagglutination” refers to clumpingof a specific subset of cells, i.e., RBCs. Thus, hemagglutination is atype of agglutination.

As used herein, the term “reduced hemagglutination” refers to detectableagglutination activity of hRBCs at anti-CD47 mAb concentrations greaterthan or equal to 1.85 μg/ml, and no measurable activity atconcentrations less than 1.85 μg/ml in a washed RBC assay, as visualizedby discrete punctate dot compared to a diffuse pattern that representshemagglutination.

As used herein, the term “no detectable hemagglutination” refers to novisible or detectable agglutination activity of hRBCs at anti-CD47 mAbconcentrations greater or equal to 0.3 pg/ml to a concentration lessthan or equal to 10 μg/ml in a washed RBC assay, as visualized bydiscrete punctate dot compared to a diffuse pattern that representshemagglutination.

Some of the anti-CD47 antibodies described herein, cause reduced or nodetectable hemagglutination of human RBCs.

Modulation of the NO Pathway

As noted above, TSP1 is also a ligand for CD47. The TSP1/CD47 pathwayopposes the beneficial effects of the NO pathway in many cell types,including, but not limited to, vascular cells. The NO pathway consistsof any of three enzymes (nitric oxide synthases, NOS I, NOS II and NOSIII) that generate bioactive gas NO using arginine as a substrate. NOcan act within the cell in which it is produced, or in neighboringcells, to activate the enzyme soluble guanylyl cyclase that produces themessenger molecule cyclic GMP (cGMP). The proper functioning of theNO/cGMP pathway is essential for protecting the cardiovascular systemagainst stresses including, but not limited to, those resulting fromwounding, inflammation, hypertension, metabolic syndrome, ischemia, andIRI. In the context of these cellular stresses the inhibition of theNO/cGMP pathway by the TSP1/CD47 system exacerbates the effects ofstress. This is a particular problem in the cardiovascular system whereboth cGMP and cAMP play important protective roles. There are many casesin which ischemia and reperfusion injury cause or contribute to disease,trauma, and poor outcomes of surgical procedures.

As disclosed herein, one of more of the chimeric or humanized anti-CD47antibodies will reverse TSP1 inhibition of cGMP production. Reversalwill be complete (>80%) or intermediate (20%-80%). This reversal of TSP1inhibition of cGMP production will demonstrate that the anti-CD47 mAbshave the ability to increase NO signaling and suggest utility inprotecting the cardiovascular system against stresses including, but notlimited to, those resulting from wounding, inflammation, hypertension,metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI).Additional assay systems, for example smooth muscle cell contraction,will also be expected to show that some of the chimeric or humanizedantibodies reverse the inhibitory actions of TSP1 on downstream effectsresulting from the activation of NO signaling.

As disclosed herein, “complete reversal of NO pathway inhibition” refersto greater than 80% reversal of TSP1 inhibition of NO signaling by ananti-CD47 mAb compared to a negative control, humanized isotype-matchedantibody or no treatment.

As disclosed herein, “intermediate reversal of NO pathway inhibition”refers to 20-80% reversal of TSP1 inhibition of NO signaling by ananti-CD47 mAb compared to a negative control, humanized isotype-matchedantibody or no treatment.

As disclosed herein, “no reversal of NO pathway inhibition” refers toless than 20% reversal of TSP1 inhibition of NO signaling by ananti-CD47 mAb compared to a negative control, humanized isotype-matchedantibody or no treatment.

Immunogenic Cell Death

The concept of immunogenic cell death (ICD) has emerged in recent years.This form of cell death, unlike non-immunogenic cell death, stimulatesan immune response against antigens from cancer cells. ICD is induced byspecific chemotherapy drugs, including anthracyclines (doxorubicin,daurorubicin and mitoxantrone) and oxaliplatin, but not by cisplatin andother chemotherapy drugs. ICD is also induced by bortezomib, cardiacglycosides, photodynamic therapy and radiation Galluzi et al. Nat RevImmunol 17: 97-111, 2016). The distinctive characteristics of ICD oftumor cells are the release from or exposure on tumor cell surfaces ofspecific ligands: 1) the pre-apoptotic cell surface exposure ofcalreticulin, 2) the secretion of adenosine triphosphate (ATP), 3)release of high mobility group box 1 (HMGB1), 4) annexin A1 release, 5)type I interferon release and 6) C-X-C motif chemokine ligand 10(CXCL10) release. These ligands are endogenous damage-associatedmolecular patterns (DAMPs), which include the cell death-associatedmolecules (CDAMs) (Kroemer et al. Annu Rev Immunol 31: 51-72, 2013).Importantly, each of these ligands induced during ICD binds to specificreceptors, referred to as pattern recognition receptors (PRRs), thatcontribute to an anti-tumor immune response. ATP binds the purinergicreceptors PY2, G-protein coupled, 2 (P2RY2) and PX2, ligand-gated ionchannel, 7 (P2RX7) on dendritic cells causing dendritic cell recruitmentand activation, respectively. Annexin A1 binds to formyl peptidereceptor 1 (FPR1) on dendritic cells causing dendritic cell homing.Calreticulin expressed on the surface of tumor cells binds to LRP1(CD91) on dendritic cells promoting antigen uptake by dendritic cells.HMGB1 binds to toll-like receptor 4 (TLR4) on dendritic cells to causedendritic cell maturation. As a component of ICD, tumor cells releasetype I interferon leading to signaling via the type I interferonreceptor and the release of the CXCL10 which favors the recruitment ofeffector CXCR3+ T cells Together, the actions of these ligands on theirreceptors facilitate recruitment of DCs into the tumor, the engulfmentof tumor antigens by DCs and optimal antigen presentation to T cells.Kroemer et al. have proposed that a precise combination of the CDAMsmentioned above elicited by ICD can overcome the mechanisms thatnormally prevent the activation of anti-tumor immune responses (Kroemeret al. Annu Rev Immunol 31: 51-72, 2013; Galluzi et al. Nat Rev Immunol17: 97-111, 2016). When mouse tumor cells treated in vitro withICD-inducing modalities are administered in vivo to syngeneic mice, theyprovide effective vaccination that leads to an anti-tumor adaptiveimmune response, including memory. This vaccination effect cannot betested in xenograft tumor models because the mice used in these studieslack a complete immune system. The available data indicate that ICDeffects induced by chemotherapy or radiation will promote an adaptiveanti-tumor immune response in cancer patients. The components of ICD aredescribed in more detail below.

In 2005, it was reported that tumor cells which were dying in responseto anthracycline chemotherapy in vitro caused an effective anti-tumorimmune response when administered in vivo in the absence of adjuvant(Casares et al. J Exp Med 202: 16911701, 2005). This immune responseprotected mice from subsequent re-challenge with viable cells of thesame tumor and caused regression of established tumors. Anthracyclines(doxorubicin, daunorubicin and idarubicin) and mitomycin C induced tumorcell apoptosis with caspase activation, but only apoptosis induced byanthracyclines resulted in immunogenic cell death. Caspase inhibitiondid not inhibit cell death induced by doxorubicin but did suppress theimmunogenicity of tumor cells dying in response to doxorubicin. Thecentral roles of dendritic cells and CD8+ T cells in the immune responseelicited by doxorubicin-treated apoptotic tumor cells were establishedby the demonstration that depletion of these cells abolished the immuneresponse in vivo.

Calreticulin is one of the most abundant proteins in the endoplasmicreticulum (ER). Calreticulin was shown to rapidly translocatepreapoptotically from the ER lumen to the surface of cancer cells inresponse to multiple ICD inducers, including anthracyclines (Obeid etal. Nat Med 13: 54-61, 2007; Kroemer et al. Annu Rev Immunol 31: 51-72,2013). Blockade or knockdown of calretiulin suppressed the phagocytosisof anthracycline-treated tumor cells by dendritic cells and abolishedtheir immunogenicity in mice. The exposure of calreticulin caused byanthracyclines or oxaliplatin is activated by an ER stress response thatinvolves the phosphorylation of the eukaryotic translation initiationfactor eIF2α by the PKR-like ER kinase. Calretiulin, which has aprominent function as an “eat-me” signal (Gardai et al. Cell 123:321-334, 2005) binds to LRP1 (CD91) on dendritic cells and macrophagesresulting in phagocytosis of the calreticulin expressing cell, unlessthe calreticulin-expressing cell expresses a don't eat me signal, suchas CD47. Calreticulin also signals through CD91 on antigen presentingcells to cause the release of proinflammatory cytokines and to programTh17 cell responses. In summary, calreticulin expressed as part ofimmunogenic cell death stimulates antigen presenting cells to engulfdying cells, process their antigens and prime an immune response.

In addition to calreticulin, protein disulfide-isomerase A3 (PDIA3),also called Erp57, was shown to translocate from the ER to the surfaceof tumor cells following treatment with mitoxantrone, oxaliplatin andirradiation with UVC light (Panaretakis et al. Cell Death Differ 15:1499-1509, 2008; Panaretakis et al. EMBL J 28: 578-590, 2009). A humanovarian cancer cell line, primary ovarian cancer cells and a humanprostate cancer cell line expressed cell-surface calreticulin, HSP70 andHSP90 following treatment with the anthracyclines doxorubicin andidarubicin (Fucikova et al. Cancer Res 71: 4821-4833, 2011). HSP70 andHSP90 bind to the PRR LRP1 on antigen presenting cells; the PRR to whichPDIA3 binds has not been identified (Galluzi et al. Nat Rev Immunol 17:97-111, 2016).

TLR4 was shown to be required for cross-presentation of dying tumorcells and to control tumor antigen processing and presentation. Amongproteins that were known to bind to and stimulate TLR4, HMGB1 wasuniquely released by mouse tumor cells in which ICD was induced byirradiation or doxorubicin (Apetoh et al. Nat Med 13: 1050-1059, 2007).The highly efficient induction of an in vivo anti-tumor immune bydoxorubicin treatment of mouse tumor cells required the presence ofHMGB1 and TLR4, as demonstrated by abrogation of the immune response byinhibition of HMGB1 and knock-out TLR4. These preclinical findings areclinically relevant. Patients with breast cancer who carry a TLR4loss-of-function allele relapse more quickly after radiotherapy andchemotherapy than those carrying the normal TLR4 allele.

Ghiringhelli et al. showed that mouse tumor cells treated withoxaliplatin, doxorubicin and mitoxanthrone in vitro released ATP andthat the ATP binds to the purinergic receptors PY2, G-protein coupled, 2(P2RY2) and PX2, ligand-gated ion channel, 7 (P2RX7) on dendritic cells(Ghiringhelli et al. Nat Med 15: 1170-1178, 2009). Binding of ATP toP2RX7 on DCs triggers the NOD-like receptor family, pyrin domaincontaining-3 protein (NLRP3)-dependent caspase-1 activation complex(inflammasome), allowing for the secretion of interleukin-10 (IL-13),which is essential for the priming of interferon-gamma-producing CD8+ Tcells by dying tumor cells. Therefore, the ATP-elicited production ofIL-10 by DCs appears to be one of the critical factors for the immunesystem to perceive cell death induced by certain chemotherapy drugs asimmunogenic. This paper also reports that HMGB1, a TLR4 agonist, alsocontributes to the stimulation of the NLRP3 inflammasome in DCs and thesecretion of IL-13. These preclinical results have been shown to haveclinical relevance; in a breast cancer cohort, the presence of the P2RX7loss-of-function allele had a significant negative prognostic impact ofmetastatic disease-free survival. ATP binding to P2RY2 causes therecruitment of myeloid cells into the tumor microenvironment (Vacchelliet al. Oncoimmunology 5: e1118600, 2016)

Michaud et al. demonstrated that autophagy is required for theimmunogenicity of chemotherapy-induced cell death (Michaud et al.Science 334: 1573-1577, 2011). Release of ATP from dying tumor cellsrequired autophagy and autophagy-competent, but not autophagy-deficient,mouse tumors attracted dendritic cells and T lymphocytes into the tumormicroenvironment in response to chemotherapy that induces ICD.

Ma et al. addressed the question of how chemotherapy-induced cell deathleads to efficient antigen presentation to T cells (Ma et al. Immunity38: 729-741, 2013). They found that at specific kind of tumorinfiltrating lymphocyte, CD11c⁺CD11b⁺Ly6C^(hi) cells, are particularlyimportant for the induction of anticancer immune responses byanthracyclines.

ATP released by dying cancer cells recruited myeloid cells into tumorsand stimulated the local differentiation of CD11c⁺CD11b⁺Ly6C^(hi) cells.These cells were shown to be particularly efficient in capturing andpresenting tumor cell antigens and, after adoptive transfer into naïvemice, conferring protection to challenge with living tumor cells of thesame cell line.

It has been shown that anthracyclines stimulate the rapid production oftype I interferons by tumor cells after activation of TLR3 (Sistugu etal. Nat Med 20: 1301-1309, 2014). Type I interferons (IFN) bind to IFN-αand IFN-β receptors on cancer cells and trigger autocrine and paracrinesignaling pathways that result in release of CXCL10. Tumors lacking Tlr3or Ifnar failed to respond to chemotherapy unless type I IFN or CXCL10,respectively, was supplied. These preclinical findings have clinicalrelevance. A type I IFN-related gene expression signature predictedclinical responses to anthracycline-based chemotherapy in independentcohorts of breast cancer patients.

Another receptor on dendritic cells that is involved inchemotherapy-induced anti-cancer immune response was recentlyidentified: formyl peptide receptor-1, which binds annexin A1 (Vacchelliet al. Science 350: 972-978, 2015). Vacchelli et al. designed a screento identify candidate genetic defects that negatively affect responsesto chemotherapy. They identified a loss-of-function allele of the geneencoding formyl peptide receptor 1 (FPR1) that was associated with poormetastatis-free survival and overall survival in breast and colorectalcancer patients receiving adjuvant chemotherapy. The therapeutic effectsof anthracyclines were abrogated in tumor-bearing Fpr1−/− mice due toimpaired antitumor immunity. FPR1-deficient DCs did not approach dyingtumor cells and, therefore, could not elicit antitumor T cell immunity.Two anthracyclines, doxorubicin and mitoxantrone, stimulated thesecretion of annexin A1, one of four known ligands of FPR1. FPR1 andannexin A1 promoted stable interactions between dying cancer cells andhuman or mouse leukocytes.

In addition to anthracyclines and oxaliplatin, other drugs have beenshown to induce immunogenic cell death. Cardiac glycosides, includingclinically used digoxin and digitoxin, were also shown to be efficientinducers of immunogenic cell death of tumor cells (Menger et al. SciTransl Med 4: 143ra99, 2012). Other chemotherapy agents and cancer drugsthat have been reported to induce DAMP expression or release arebleomycin, bortezomib, cyclophosphamide, paclitaxel, vorinistat andcisplatin (Garg et al. Front Immunol 588: 1-24, 2015; Menger et al. SciTransl Med 4: 143ra99, 2012; Martins et al. Oncogene 30: 1147-1158,2011). Importantly, these results have clinical relevance.Administration of digoxin during chemotherapy had a significant positiveimpact on the overall survival of patients with breast, colorectal, headand neck, and hepatocellular cancers, but failed to improve overallsurvival of lung and prostate cancer patients.

The anti-CD20 monoclonal antibody rituximab has improved outcomes inmultiple B-cell malignancies. The success of rituximab, referred to as atype I anti-CD20 mAb, led to the development of type II anti-CD20 mAbs,including obinutuzumab and tositumomab. Cheadle et al., investigated theinduction of immunogenic cell death by anti-CD20 mAbs (Cheadle et al.Brit J Haematol 162: 842-862, 2013). They found that the cell deathinduced by obinutuzumab and tositumomab is a form of immunogenic celldeath characterized by the release of HMGB1, HSP90 and ATP. A type Ianti-CD20 mAb did not cause release of HMGB1, HSP90 and ATP. Incubationof supernatants from a human tumor cell line treated with obinutuzumabcaused maturation of human dendritic cells, consistent with thepreviously described effects of HMGB1 and ATP on dendritic cells. Incontrast to the results reported by Cheadle et al., Zhao et al. reportedthat both type I and II anti-CD20 mAbs increased HMGB1 release fromhuman diffuse large B cell lymphoma cell lines, but did not cause ATPrelease or cell surface expression of calreticulin (Zhao et al.Oncotarget 6: 27817-27831, 2015).

The release from or exposure on tumor cell surfaces of the DAMPscalreticulin, ATP, HMGB1, annexin A1, type I interferon release, CXCL10,PDIA3, HSP70 and/or HSP90 in response to anti-CD47 mAbs has not beenreported. As disclosed herein, anti-CD47 mAbs cause release from orexposure on tumor cell surfaces of one or more of the DAMPs listed above(characteristics of ICD), an unexpected result. These DAMPS are expectedto promote a therapeutically beneficial adaptive anti-tumor immuneresponse.

As disclosed herein, “causes an increase in cell surface calreticulinexpression on human tumor cells” refers to a statistically significantincrease (p<0.05) in calreticulin expression by a soluble anti-CD47 mAbcompared to the background obtained with a negative control, humanizedisotype-matched antibody or no treatment.

As disclosed herein, the term “the release of” is synonymous withsecretion and is defined as the extracellular appearance of ATP, HMGB1,annexin A1, type I interferon and CXCL10.

As disclosed herein, “cause an increase in the release of adenosinetriphosphate by human tumor cells” refers to a statistically significantincrease (p<0.05) in ATP in the supernatant caused by a solubleanti-CD47 mAb compared to the background obtained with a negativecontrol, humanized isotype-matched antibody or no treatment.

As disclosed herein, “cause an increase in the release of high mobilitygroup box 1 by human tumor cells” refers to a statistically significantincrease (p<0.05) in HMGB1 in the supernatant caused by a solubleanti-CD47 mAb compared to the background obtained with a negativecontrol, humanized isotype-matched antibody or no treatment.

As disclosed herein, “causes an increase in the release of type Iinterferon by human tumor cells” refers to a statistically significantincrease (p<0.05) in type I interferon in the supernatant or type Iinterferon mRNA caused by a soluble anti-CD47 mAb compared to thebackground obtained with a negative control, humanized isotype-matchedantibody or no treatment.

As disclosed herein, “causes an increase in the release of C-X-C MotifChemokine Ligand 10 (CXCL10) by human tumor cells” refers to astatistically significant increase (p<0.05) in CXCL10 in the supernatantor CXCL10 mRNA caused by a soluble anti-CD47 mAb compared to thebackground obtained with a negative control, humanized isotype-matchedantibody or no treatment.

As disclosed herein, “causes an increase in cell surface PDIA3expression on human tumor cells” refers to a statistically significantincrease (p<0.05) in PDIA3 expression by a soluble anti-CD47 mAbcompared to the background obtained with a negative control, humanizedisotype-matched antibody or no treatment.

As disclosed herein, “causes an increase in cell surface HSP70expression on human tumor cells” refers to a statistically significantincrease (p<0.05) in HSP70 expression by a soluble anti-CD47 mAbcompared to the background obtained with a negative control, humanizedisotype-matched antibody or no treatment.

As disclosed herein, “causes an increase in cell surface HSP90expression on human tumor cells” refers to statistically significantincrease (p<0.05) in HSP90 expression by a soluble anti-CD47 mAbcompared to the background obtained with a negative control, humanizedisotype-matched antibody or no treatment.

pH Dependence of Anti-CD47 mAb Binding

Most antibody binding, particularly in the blood compartment and tonormal cells occurs at physiological pH (approximately 7.4). Therefore,the binding affinity of therapeutic mAbs is normally assessed in vitroat physiological pH. However, the tumor microenvironment (TME) is moreacidic in nature, with pH values below 7.4. This appears to be due to anumber of differences including hypoxia, anaerobic glycolysis leading tothe production of lactic acid and hydrolysis of ATP (Tannock and Rotin,Cancer Res 1989; Song et al., Cancer Drug Discovery and Development2006; Chen and Pagel, Advan Radiol 2015). The acidic pH may provide anadvantage to the tumor by promoting invasiveness, metastatic behavior,chronic autophagy, resistance to chemotherapies and reduced efficacy ofimmune cells in the tumor microenvironment (Estrella et al. Cancer Res2013; Wojtkowiak et al., Cancer Res 2012; Song et al., Cancer DrugDiscovery and Development 2006; Barar, BioImpacts, 2012). Theidentification of anti-CD47 antibodies with the property of increasedbinding affinity at acidic pH would confer a therapeutic advantage withhigher binding to CD47 on tumor cells within the acidic TME compared tocells at physiological pH. Antibodies with pH-dependent properties havebeen generated with the goal of recycling antibodies. However, incontrast to exhibiting the properties of enhanced binding at acidic pH,these bind with high affinity to their target antigen at physiologicalpH, but release their target at acidic pH (Bonvin et al., mAbs 2015;Igawa and Hattori, Biochem Biophys Acta 2014).

As disclosed herein, “has a greater affinity for CD47 at an acidic pHcompared to physiological pH” refers to an apparent Kd that is increased5-fold or more at acidic pH (<7.4) compared to physiological pH (7.4).

Combinations of Functional Properties

In some embodiments, the anti-CD47 antibodies described herein, are alsocharacterized by combinations of properties which are not exhibited byprior art anti-CD47 antibodies proposed for human therapeutic use.Accordingly, in some embodiments, anti-CD47 antibodies described hereinmay be characterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells; and    -   d. induces death of susceptible human tumor cells.

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. induces death of susceptible human tumor cells; and    -   e. causes no detectable agglutination of human red blood cells        (hRBCs).

In yet another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. induces death of susceptible human tumor cells; and    -   e. causes reduced agglutination of human red blood cells        (hRBCs).

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. induces death of susceptible human tumor cells; and    -   e. has reduced hRBC binding.

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. causes no detectable agglutination of human red blood cells        (hRBCs); and    -   e. has minimal binding to hRBCs.

In another embodiment described herein, the anti-CD47 antibodies arecharacterized by:

-   -   a. binds to human CD47;    -   b. blocks SIRPα binding to human CD47;    -   c. increases phagocytosis of human tumor cells;    -   d. causes no detectable agglutination of human red blood cells        (hRBCs); and    -   e. has reduced hRBC binding.

In another embodiment described herein, the monoclonal antibody, orantigen binding fragment thereof binds to human, non-human primate,mouse, rabbit, and rat CD47.

In yet another embodiment described herein, the monoclonal antibody, orantigen binding fragment thereof specifically also binds to non-humanprimate CD47, wherein non-human primate may include, but is not limitedto, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.

In another embodiment, the anti-CD47 monoclonal antibody, or antigenbinding fragment thereof, may additionally possess one or more of thefollowing characteristics: 1) exhibit cross-reactivity with one or morespecies homologs of CD47; 2) block the interaction between CD47 and itsligand SIRPα; 3) increase phagocytosis of human tumor cells; 4) inducedeath of susceptible human tumor cells; 5) do not induce cell death ofhuman tumor cells; 6) do not have reduced or minimal binding to humanred blood cells (hRBCs); 7) have reduced binding to hRBCs; 8) haveminimal binding to hRBCs; 9) cause reduced agglutination of hRBCs; 10)cause no detectable agglutination of hRBCs; 11) reverse TSP1 inhibitionof the nitric oxide (NO) pathway; 12) do not reverse TSP1 inhibition ofthe NO pathway; 13) cause loss of mitochondrial membrane potential; 14)do not cause cause loss of mitochondrial membrane potential; 15) causean increase in cell surface calreticulin expression on human tumorcells; 16) do not cause an increase in cell surface calreticulinexpression on human tumor cells; 17) cause an increase in adenosinetriphosphate (ATP) release by human tumor cells; 18) do not cause anincrease in adenosine triphosphate (ATP) release by human tumor cells;19) cause an increase in high mobility group box 1 (HMGB1) release byhuman tumor cells; 20) do not cause an increase in high mobility groupbox 1 (HMGB1) release by human tumor cells; 21) cause an increase intype I interferon release by human tumor cells; 22) do not cause anincrease in type I interferon release by human tumor cells; 23) cause anincrease in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by humantumor cells; 24) do not cause an increase in C-X-C Motif ChemokineLigand 10 (CXCL10) release by human tumor cells; 25) cause an increasein cell surface protein disulfide-isomerase A3 (PDIA3) expression onhuman tumor cells; 26) do not cause an increase in cell surface proteindisulfide-isomerase A3 (PDIA3) expression on human tumor cells; 27)cause an increase in cell surface heat shock protein 70 (HSP70)expression on human tumor cells; 28) do not cause an increase in cellsurface heat shock protein 70 (HSP70) expression on human tumor cells;29) cause an increase in cell surface heat shock protein 90 (HSP90)expression on human tumor cells; 30) do not cause an increase in cellsurface heat shock protein 90 (HSP90) expression on human tumor cells;31) have reduced binding to normal human cells, which includes, but isnot limited to, endothelial cells, skeletal muscle cells, epithelialcells, and peripheral blood mononuclear cells (e.g., human aorticendothelial cells, human skeletal muscle cells, human microvascularendothelial cells, human renal tubular epithelial cells, humanperipherial blood CD3+ cells, and human peripheral blood mononuclearcells); 32) do not have reduced binding to normal human cells, whichincludes, but is not limited to, endothelial cells, skeletal musclecells, epithelial cells, and peripheral blood mononuclear cells (e.g.,human aortic endothelial cells, human skeletal muscle cells, humanmicrovascular endothelial cells, human renal tubular epithelial cells,human peripherial blood CD3+ cells, and human peripheral bloodmononuclear cells); 33) have a greater affinity for human CD47 at anacidic pH compared to physiological pH; 34) do not have a greateraffinity for human CD47 at an acidic pH compared to physiological pH;and 35) cause an increase in annexin A1 release by human tumor cells.

In some embodiments, a monoclonal antibody, or an antigen bindingfragment thereof, is provided, which: binds to human CD47; blocks SIRPαbinding to human CD47; increases phagocytosis of human tumor cells; andinduces death of human tumor cells; wherein said monoclonal antibody, oran antigen binding fragment thereof, exhibits pH-dependent binding toCD47 present on a cell. In other embodiments, the disclosure provides amonoclonal antibody, or an antigen binding fragment thereof, which:binds to human CD47; blocks SIRPα binding to human CD47; increasesphagocytosis of human tumor cells; and induces death of human tumorcells; wherein said monoclonal antibody, or an antigen binding fragmentthereof, exhibits reduced binding to normal cells. In some embodiments,a cell to which such an antibody may bind may be of any cell type asdescribed herein. In other embodiments, a monoclonal antibody asdescribed herein, or an antigen binding fragment thereof, may exhibitany combination of characteristics provided in the present disclosure.For example, a monoclonal antibody may beneficially exhibit both pHdependent binding and reduced binding to a cell. These cells may be anendothelial cell, a skeletal muscle cell, an epithelial cell, a PBMC ora RBC (e.g., human aortic endothelial cells, human skeletal musclecells, human microvascular endothelial cells, human renal tubularepithelial cells, human peripherial blood CD3+ cells, human peripheralblood mononuclear cells or human RBC). Such characteristics may beexhibited individually or in any combination as described herein. Asused herein, pH dependent binding of an antibody of the disclosure mayrefer to altered binding of the antibody at a particular pH, for examplean antibody that exhibits increased binding affinity at acidic pH.

CD47 Antibodies

Many human cancers up-regulate cell surface expression of CD47 and thoseexpressing the highest levels of CD47 appear to be the most aggressiveand the most lethal for patients. Increased CD47 expression is thoughtto protect cancer cells from phagocytic clearance by sending a “don'teat me” signal to macrophages via SIRPα, an inhibitory receptor thatprevents phagocytosis of CD47-bearing cells (Oldenborg et al. Science288: 2051-2054, 2000; Jaiswal et al. (2009) Cell 138(2):271-851; Chao etal. (2010) Science Translational Medicine 2(63):63ra94). Thus, theincrease of CD47 expression by many cancers provides them with a cloakof “selfness” that slows their phagocytic clearance by macrophages anddendritic cells.

Antibodies that block CD47 and prevent its binding to SIRPα have shownefficacy in human tumor in murine (xenograft) tumor models. Suchblocking anti-CD47 mAbs exhibiting this property increase thephagocytosis of cancer cells by macrophages, which can reduce tumorburden (Majeti et al. (2009) Cell 138 (2): 286-99; U.S. Pat. No.9,045,541; Willingham et al. (2012) Proc Natl Acad. Sci. USA109(17):6662-6667; Xiao et al. (2015) Cancer Letters 360:302-309; Chaoet al. (2012) Cell 142:699-713; Kim et al. (2012) Leukemia26:2538-2545).

Anti-CD47 mAbs have also been shown to promote an adaptive immuneresponse to tumors in vivo (Tseng et al. (2013) PNAS 110(27):11103-11108; Soto-Pantoja et al. (2014) Cancer Res. 74 (23):6771-6783; Liu et al. (2015) Nat. Med. 21 (10): 1209-1215; Xu et al.(2017) Immunity 47: 363-373).

However, there are mechanisms by which anti-CD47 mAbs can attacktransformed cells that have not yet been exploited in the treatment ofcancer. Multiple groups have shown that particular anti-human CD47 mAbsinduce cell death of human tumor cells. Anti-CD47 mAb Ad22 induces celldeath of multiple human tumor cells lines (Pettersen et al. J. Immunol.166: 4931-4942, 2001; Lamy et al. J. Biol. Chem. 278: 23915-23921,2003). AD22 was shown to indice rapid mitochondrial dysfunction andrapid cell death with early phosphatidylserine exposure and a drop inmitochondrial membrane potential (Lamy et al. J. Biol. Chem. 278:23915-23921, 2003). Anti-CD47 mAb MABL-2 and fragments thereof inducecell death of human leukemia cell lines, but not normal cells in vitroand had an anti-tumor effect in in vivo xenograft models. (Uno et al.(2007) Oncol. Rep. 17 (5): 1189-94). Anti-human CD47 mAb 1F7 inducescell death of human T cell leukemias (Manna and Frazier (2003) J.Immunol. 170: 3544-53) and several breast cancers (Manna and Frazier(2004) Cancer Research 64 (3):1026-36). 1F7 kills CD47-bearing tumorcells without the action of complement or cell mediated killing by NKcells, T cells, or macrophages. Instead, anti-CD47 mAb 1F7 acts via anon-apoptotic mechanism that involves a direct CD47-dependent attack onmitochondria, discharging their membrane potential and destroying theATP-generating capacity of the cell leading to rapid cell death. It isnoteworthy that anti-CD47 mAb 1F7 does not kill resting leukocytes,which also express CD47, but only those cells that are “activated” bytransformation. Thus, normal circulating cells, many of which expressCD47, are spared while cancer cells are selectively killed by thetumor-toxic CD47 mAb (Manna and Frazier (2003) J. Immunol. 170:3544-53). This mechanism can be thought of as a proactive, selective anddirect attack on tumor cells in contrast to the passive mechanism ofcausing phagocytosis by simply blocking CD47/SIRPα binding. Importantly,mAb 1F7 also blocks binding of SIRPα to CD47 (Rebres et al., J. CellularPhysiol. 205: 182-193, 2005) and thus it can act via two mechanisms: (1)direct tumor toxicity, and (2) causing phagocytosis of cancer cells. Asingle mAb that can accomplish both functions may be superior to onethat only blocks CD47/SIRPα binding.

An additional mechanism by which anti-CD47 mAbs can be exploited in thetreatment of cancer is through the promotion of an anti-tumor immuneresponse. The discovery that anti-CD47 mAbs cause tumor cells to releaseDAMPs that cause maturation, activation and homing of DCs and attractionof T cells connects anti-CD47 mAb treatment to the development of thetherapeutically desirable anti-tumor immune response. Anti-CD47 mAbshave not been previously shown to cause tumor cell release of ATP,HMGB1, annexin A1, type I interferons and CXCL10 and tumor cellexpression of calreticulin, PDIA3, HSP70 and HSP90.

Following periods of tissue ischemia, the initiation of blood flowcauses damage referred to as “ischemia-reperfusion injury” or IRI. IRIcontributes to poor outcomes in many surgical procedures where IRIoccurs due to the necessity to stop blood flow for a period of time, inmany forms/causes of trauma in which blood flow is interrupted and laterrestored by therapeutic intervention and in procedures required fororgan transplantation, cardio/pulmonary bypass procedures, reattachmentof severed body parts, reconstructive and cosmetic surgeries and othersituations involving stopping and restarting blood flow. Ischemia itselfcauses many physiological changes that, by themselves would eventuallylead to cell and tissue necrosis and death. Reperfusion poses its ownset of damaging events including generation of reactive oxygen species,thrombosis, inflammation and cytokine mediated damage. The pathways thatare limited by the TSP1-CD47 system are precisely those that would be ofmost benefit in combating the damage of IRI, including the NO pathway.Thus, blocking the TSP1-CD47 pathway, as with the antibodies disclosedherein, will provide more robust functioning of these endogenousprotective pathways. Anti-CD47 mAbs have been shown to reduce organdamage in rodent models of renal warm ishchemia (Rogers et al. J Am SocNephrol. 23: 1538-1550, 2012), liver ischemia-reperfusion injury(Isenberg et al. Surgery. 144: 752-761, 2008), renal transplantation(Lin et al. Transplantation. 98: 394-401, 2014; Rogers et al. KidneyInterantional. 90: 334-347, 2016)) and liver transplantation, includingsteatotic livers (Xiao et al. Liver Transpl. 21: 468-477, 2015; Xiao etal. Transplantation. 100: 1480-1489, 2016). In addition, anti-CD47 mAbcaused significant reductions of right ventricular systolic pressure andright ventricular hypertrophy in the monocrotaline model of pulmonaryarterial hypertension (Bauer et al. Cardiovasc Res. 93: 682-693, 2012).Studies in skin flap models have shown that modulation of CD47,including with anti-CD47 mAbs, inhibits TSP1-mediated CD47 signaling.This results in inceased activity of the NO pathway, resulting inreduced IRI (Maxhimer et al. Plast Reconstr Surg. 124: 1880-1889, 2009;Isenberg et al. Arterioscler Throm Vasc Biol. 27: 2582-2588, 2007;Isenberg et al. Curr Drug Targets. 9: 833-841, 2008; Isenberg et al. AnnSurg. 247: 180-190, 2008) Anti-CD47 mAbs have also been shown to beefficacious in models of other cardiovascular diseases. In the mousetransverse aortic constriction model of pressure overload leftventricular heart failure, anti-CD47 mAb mitigated cardiac myocytehypertrophy, decreased left ventricular fibrosis, prevented an increasein left ventricular weight, decreased ventricular stiffness, andnormalized changes in the pressure volume loop profile (Sharifi-Sanjaniet al. J Am Heart Assoc., 2014). An anti-CD47 mAb amelioratedatherosclerosis in multiple mouse models (Kojima et al. Nature., 2016).

Cancer Indications

Presently disclosed are anti-CD47 mAbs and antigen binding fragmentsthereof effective as cancer therapeutics which can be administered topatients, preferably parenterally, with susceptible hematologic cancersand solid tumors including, but not limited to, leukemias, includingsystemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL),T cell—ALL, acute myeloid leukemia (AML), myelogenous leukemia, chroniclymphocytic leukemia (CLL), chronic myeloid leukemia (CML),myeloproliferative disorder/neoplasm, monocytic cell leukemia, andplasma cell leukemia; multiple myeloma (MM); Waldenstrom'sMacroglobulinemia; lymphomas, including histiocytic lymphoma and T celllymphoma, B cell lymphomas, including Hodgkin's lymphoma andnon-Hodgkin's lymphoma, such as low grade/follicular non-Hodgkin'slymphoma (NHL), cell lymphoma (FCC), mantle cell lymphoma (MCL), diffuselarge cell lymphoma (DLCL), small lymphocytic (SL) NHL, intermediategrade/follicular NHL, intermediate grade diffuse NHL, high gradeimmunoblastic NHL, high grade lymphoblastic NHL, high grade smallnon-cleaved cell NHL, bulky disease NHL; solid tumors, including ovariancancer, breast cancer, endometrial cancer, colon cancer (colorectalcancer), rectal cancer, bladder cancer, urothelial cancer, lung cancer(non-small cell lung cancer, adenocarcinoma of the lung, squamous cellcarcinoma of the lung), bronchial cancer, bone cancer, prostate cancer,pancreatic cancer, gastric cancer, hepatocellular carcinoma (livercancer, hepatoma), gall bladder cancer, bile duct cancer, esophagealcancer, renal cell carcinoma, thyroid cancer, squamous cell carcinoma ofthe head and neck (head and neck cancer), testicular cancer, cancer ofthe endocrine gland, cancer of the adrenal gland, cancer of thepituitary gland, cancer of the skin, cancer of soft tissues, cancer ofblood vessels, cancer of brain, cancer of nerves, cancer of eyes, cancerof meninges, cancer of oropharynx, cancer of hypopharynx, cancer ofcervix, and cancer of uterus, glioblastoma, meduloblastoma, astrocytoma,glioma, meningioma, gastrinoma, neuroblastoma, myelodysplastic syndrome,and sarcomas including, but not limited to, osteosarcoma, Ewing'ssarcoma, leiomyosarcoma, synovial sarcoma, alveolar soft part sarcoma,angiosarcoma, liposarcoma, fibrosarcoma, rhabdomyosarcoma,chrondrosarcoma, and melanoma.

Treatment of Cancer

As is well known to those of ordinary skill in the art, combinationtherapies are often employed in cancer treatment as single-agenttherapies or procedures may not be sufficient to treat or cure thedisease or condition. Conventional cancer treatments often involvesurgery, radiation treatment, the administration of a combination ofcytotoxic drugs to achieve additive or synergistic effects, andcombinations of any or all of these approaches. Especially usefulchemotherapeutic and biologic therapy combinations employ drugs thatwork via different mechanisms of action, increasing cancer cell controlor killing, increasing the ability of the immune system to controlcancer cell growth, reducing the likelihood of drug resistance duringtherapy, and minimizing possible overlapping toxicities by permittingthe use of reduced doses of individual drugs.

Classes of conventional anti-tumor/anti-neoplastic agents useful in thecombination therapies encompassed by the present methods are disclosed,for example, in Goodman & Gilman's The Pharmacological Basis ofTherapeutics, Twelfth Edition (2010) L. L. Brunton, B. A. Chabner, andB. C. Knollmann Eds., Section VIII, “Chemotherapy of NeoplasticDiseases”, Chapters 60-63, pp. 1665-1770, McGraw-Hill, NY, and include,for example, alkylating agents, antimetabolites, natural products, avariety of miscellaneous agents, hormones and antagonists, targeteddrugs, monoclonal antibodies and other protein therapeutics.

In addition to the foregoing, the methods of the present disclosure arerelated to treatment of cancer indications and further comprisestreating the patient via surgery, radiation, and/or administering to apatient in need thereof an effective amount of a chemical small moleculeor biologic drug including, but not limited to, a peptide, polypeptide,protein, nucleic acid therapeutic, conventionally used or currentlybeing developed, to treat tumorous conditions. This includes antibodiesand antigen-binding fragments, other than those disclosed herein,cytokines, antisense oligonucleotides, siRNAs, and miRNAs.

The therapeutic methods disclosed and claimed herein include the use ofthe antibodies disclosed herein alone, and/or in combinations with oneanother, and/or with antigen-binding fragments thereof of the presentdisclosure that bind to CD47, and/or with competing antibodiesexhibiting appropriate biological/therapeutic activity, as well, forexample, all possible combinations of these antibody compounds toachieve the greatest treatment efficacy.

In addition, the present therapeutic methods also encompass the use ofthese antibodies, antigen-binding fragments thereof, competingantibodies and combinations thereof further in combination with: (1) anyone or more anti-tumor therapeutic treatments selected from surgery,radiation, anti-tumor, anti-neoplastic agents, and combinations of anyof these, or (2) any one or more of anti-tumor biological agents, or (3)equivalents of any of the foregoing of (1) or (2) as would be apparentto one of ordinary skill in the art, in appropriate combination(s) toachieve the desired therapeutic treatment effect for the particularindication.

Antibody and small molecule drugs that increase the immune response tocancer by modulating co-stimulatory or inhibitory interactions thatinfluence the T cell response to tumor antigens, including inhibitors ofimmune checkpoints and modulators of co-stimulatory molecules, are alsoof particular interest in the context of the combination therapeuticmethods encompassed herein and include, but are not limited to, otheranti-CD47 antibodies. Administration of therapeutic agents that bind tothe CD47 protein, for example, antibodies or small molecules that bindto CD47 and prevent interaction between CD47 and SIRPα, are administeredto a patient, causing the clearance of cancer cells via phagocytosis.The therapeutic agent that binds to the CD47 protein is combined with atherapeutic agent such as an antibody, a chemical small molecule orbiologic drug disclosed herein, directed against one or more additionalcellular targets of CD70 (Cluster of Differentiation 70), CD200 (OX-2membrane glycoprotein, Cluster of Differentiation 200), CD154 (Clusterof Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation40 ligand), CD223 (Lymphocyte-activation gene 3, LAG3, Cluster ofDifferentiation 223), KIR (Killer-cell immunoglobulin-like receptors),GITR (TNFRSF18, glucocorticoid-induced TNFR-related protein,activation-inducible TNFR family receptor, AITR, Tumor necrosis factorreceptor superfamily member 18), CD28 (Cluster of Differentiation 28),CD40 (Cluster of Differentiation 40, Bp50, CDW40, TNFRSF5, Tumornecrosis factor receptor superfamily member 5, p50), CD86 (B7-2, Clusterof Differentiation 86), CD160 (Cluster of Differentiation 160, BY55,NK1, NK28), CD258 (LIGHT, Cluster of Differentiation 258, Tumor necrosisfactor ligand superfamily member 14, TNFSF14, HVEML, HVEM ligand,herpesvirus entry mediator ligand, LTg), CD270 (HVEM, Tumor necrosisfactor receptor superfamily member 14, herpesvirus entry mediator,Cluster of Differentiation 270, LIGHTR, HVEA), CD275 (ICOSL, ICOSligand, Inducible T-cell co-stimulator ligand, Cluster ofDifferentiation 275), CD276 (B7-H3, B7 homolog 3, Cluster ofDifferentiation 276), OX40L (OX40 Ligand), B7-H4 (B7 homolog 4, VTCN1,V-set domain-containing T-cell activation inhibitor 1), GITRL(Glucocorticoid-induced tumor necrosis factor receptor-ligand,glucocorticoid-induced TNFR-ligand), 4-1BBL (4-1BB ligand), CD3 (Clusterof Differentiation 3, T3D), CD25 (IL2Ru, Cluster of Differentiation 25,Interleukin-2 Receptor a chain, IL-2 Receptor a chain), CD48 (Cluster ofDifferentiation 48, B-lymphocyte activation marker, BLAST-1, signalinglymphocytic activation molecule 2, SLAMF2), CD66a (Ceacam-1,Carcinoembryonic antigen-related cell adhesion molecule 1, biliaryglycoprotein, BGP, BGP1, BGPI, Cluster of Differentiation 66a), CD80(B7-1, Cluster of Differentiation 80), CD94 (Cluster of Differentiation94), NKG2A (Natural killer group 2A, killer cell lectin-like receptorsubfamily D member 1, KLRD1), CD96 (Cluster of Differentiation 96,TActILE, T cell activation increased late expression), CD112 (PVRL2,nectin, Poliovirus receptor-related 2, herpesvirus entry mediator B,HVEB, nectin-2, Cluster of Differentiation 112), CD115 (CSF1R, Colonystimulating factor 1 receptor, macrophage colony-stimulating factorreceptor, M-CSFR, Cluster of Differentiation 115), CD205 (DEC-205, LY75,Lymphocyte antigen 75, Cluster of Differentiation 205), CD226 (DNAM1,Cluster of Differentiation 226, DNAX Accessory Molecule-1, PTA1,platelet and T cell activation antigen 1), CD244 (Cluster ofDifferentiation 244, Natural killer cell receptor 2B4), CD262 (DR5,TrailR2, TRAIL-R2, Tumor necrosis factor receptor superfamily member10b, TNFRSF10B, Cluster of Differentiation 262, KILLER, TRICK2, TRICKB,ZTNFR9, TRICK2A, TRICK2B), CD284 (Toll-like Receptor-4, TLR4, Cluster ofDifferentiation 284), CD288 (Toll-like Receptor-8, TLR8, Cluster ofDifferentiation 288), TNFSF15 (Tumor necrosis factor superfamily member15, Vascular endothelial growth inhibitor, VEGI, TL1A), TDO2 (Tryptophan2,3-dioxygenase, TPH2, TRPO), IGF-1R (Type I Insulin-like GrowthFactor), GD2 (Disialoganglioside 2), TMIGD2 (Transmembrane andimmunoglobulin domain-containing protein 2), RGMB (RGM domain family,member B), VISTA (V-domain immunoglobulin-containing suppressor ofT-cell activation, B7-H5, B7 homolog 5), BTNL2 (Butyrophilin-likeprotein 2), Btn (Butyrophilin family), TIGIT (T cell Immunoreceptor withIg and ITIM domains, Vstm3, WUCAM), Siglecs (Sialic acid binding Ig-likelectins), Neurophilin, VEGFR (Vascular endothelial growth factorreceptor), ILT family (LIRs, immunoglobulin-like transcript family,leukocyte immunoglobulin-like receptors), NKG families (Natural killergroup families, C-type lectin transmembrane receptors), MICA (MHC classI polypeptide-related sequence A), TGFβ (Transforming growth factor β),STING pathway (Stimulator of interferon gene pathway), Arginase(Arginine amidinase, canavanase, L-arginase, arginine transamidinase),EGFRvIII (Epidermal growth factor receptor variant III), and HHLA2(B7-H7, B7y, HERV-H LTR-associating protein 2, B7 homolog 7), inhibitorsof PD-1 (Programmed cell death protein 1, PD-1, CD279, Cluster ofDifferentiation 279), PD-L1 (B7-H1, B7 homolog 1, Programmeddeath-ligand 1, CD274, cluster of Differentiation 274), PD-L2 (B7-DC,Programmed cell death 1 ligand 2, PDCD1LG2, CD273, Cluster ofDifferentiation 273), CTLA-4 (Cytotoxic T-lymphocyte-associated protein4, CD152, Cluster of Differentiation 152), BTLA (B- and T-lymphocyteattenuator, CD272, Cluster of Differentiation 272), Indoleamine2,3-dioxygenase (IDO, IDO1), TIM3 (HAVCR2, Hepatitis A virus cellularreceptor 2, T cell immunoglobulin mucin-3, KIM-3, Kidney injury molecule3, TIMD-3, T cell immunoglobulin mucin-domain 3), A2A adenosine receptor(ADO receptor), CD39 (ectonucleoside triphosphate diphosphohydrolase-1,Cluster of Differentiation 39, ENTPDi), and CD73 (Ecto-5′-nucleotidase,5′-nucleotidase, 5′-NT, Cluster of Differentiation 73), CD27 (Cluster ofDifferentiation 27), ICOS (CD278, Cluster of Differentiation 278,Inducible T-cell Co-stimulator), CD137 (4-1BB, Cluster ofDifferentiation 137, tumor necrosis factor receptor superfamily member9, TNFRSF9), OX40 (CD134, Cluster of Differentiation 134), and TNFSF25(Tumor necrosis factor receptor superfamily member 25), includingantibodies, small molecules, and agonists, are also specificallycontemplated herein. Additional agents include IL-10 (Interleukin-10,human cytokine synthesis inhibitory factor, CSIF) and Galectins.

YERVOY® (ipilimumab; Bristol-Meyers Squibb) is an example of an approvedanti-CTLA-4 antibody.

KEYTRUDA® (pembrolizumab; Merck) and OPDIVO® (nivolumab; Bristol-MeyersSquibb Company) are examples of approved anti-PD-1 antibodies.

TECENTRIQ® (atezolizumab; Roche) is an example of an approved anti-PD-L1antibody.

Ischemia-Reperfusion Injury (IRI)-Related, Autoimmune, Autoinflammatory,Inflammatory, Cardiovascular Conditions and Diseases

Administration of a CD47 mAb or antigen binding fragment thereofdisclosed herein can be used to treat a number of diseases andconditions in which IRI is a contributing feature, and to treat variousautoimmune, autoinflammatory, inflammatory and cardiovascular diseases.These include: organ transplantation in which a mAb or antigen bindingfragment thereof of the present disclosure is administered to the donorprior to organ harvest, to the harvested donor organ in the organpreservation solution, to the recipient patient, or to any combinationthereof; skin grafting; surgical resections or tissue reconstruction inwhich such mAb or fragment is administered either locally by injectionto the affected tissue or parenterally to the patient; reattachment ofbody parts; treatment of traumatic injury; pulmonary hypertension;pulmonary arterial hypertension; sickle cell disease (crisis);myocardial infarction; cerebrovascular disease; stroke;surgically-induced ischemia; acute kidney disease/kidney failure; anyother condition in which IRI occurs and contributes to the pathogenesisof disease; autoimmune and inflammatory diseases, including arthritis,rheumatoid arthritis, multiple sclerosis, psoriasis, psoriaticarthritis, Crohn's disease, inflammatory bowel disease, ulcerativecolitis, lupus, systemic lupus erythematous, juvenile rheumatoidarthritis, juvenile idiopathic arthritis, Grave's disease, Hashimoto'sthyroiditis, Addison's disease, celiac disease, dermatomyositis,multiple sclerosis, myasthenia gravis, pernicious anemia, Sjögrensyndrome, type I diabetes, vasculitis, uveitis, and ankylosingspondylitis; autoinflammatory diseases, including familial Mediterraneanfever, neonatal onset multisystem inflammatory disease, tumor necrosisfactor (TNF) receptor-associated periodic syndrome, deficiency of theinterleukin-1 receptor antagonist, Behget's disease; cardiovasculardiseases, including coronary heart disease, coronary artery disease,atherosclerosis, myocardial infarction, heart failure, and leftventricular heart failure.

Anti-CD47 mAbs and antigen binding fragments thereof of the presentdisclosure can also be used to increase tissue perfusion in a subject inneed of such treatment. Such subjects can be identified by diagnosticprocedures indicating a need for increased tissue perfusion. Inaddition, the need for increased tissue perfusion may arise because thesubject has had, is having, or will have, a surgery selected fromintegument surgery, soft tissue surgery, composite tissue surgery, skingraft surgery, resection of a solid organ, organ transplant surgery, orreattachment or an appendage or other body part.

Treatment of Ischemia-Reperfusion Injury (IRI)-Related Indications

The methods of the present disclosure, for example those related totreatment of IRI-related indications, can further comprise administeringto a patient in need thereof an effective amount of therapeutic agentthat binds to the CD47 protein and a nitric oxide donor, precursor, orboth; a nitric oxide generating topical agent; an agent that activatessoluble guanylyl cyclase; an agent that inhibits cyclic nucleotidephosphodiesterases; or any combination of any of the foregoing.

In these methods, the nitric oxide donor or precursor can be selectedfrom NO gas, isosorbide dinitrate, nitrite, nitroprusside,nitroglycerin, 3-Morpholinosydnonimine (SIN-1),S-nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO),S-nitrosothiols, Bidil®, and arginine.

The agent that activates soluble guanylyl cyclase can be a non-NO(nitric oxide)-based chemical activator of soluble guanylyl cyclase thatincreases cGMP levels in vascular cells. Such agents bind solubleguanylyl cyclase in a region other than the NO binding motif, andactivate the enzyme regardless of local NO or reactive oxygen stress(ROS). Non-limiting examples of chemical activators of soluble guanylylcyclase include organic nitrates (Artz et al. (2002) J. Biol. Chem.277:18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl.Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood84:4226-4233); BAY 41-2272 and BAY 41-8543 (Stasch et al. (2001 Nature410 (6825): 212-5), CMF-1571, and A-350619 (reviewed in Evgenov et al.(2006) Nat. Rev. Drug. Discov. 5:755-768); BAY 58-2667 (Cinaciguat; Freyet al. (2008) Journal of Clinical Pharmacology 48 (12): 1400-10); BAY63-2521 (Riociguat; Mittendorf et al. (2009) Chemmedchem 4 (5): 853-65).Additional soluble guanylyl cyclase activators are disclosed in Staschet al. (2011) Circulation 123:2263-2273; Derbyshire and Marletta (2012)Ann. Rev. Biochem. 81:533-559, and Nossaman et al. (2012) Critical CareResearch and Practice, Volume 2012, Article ID 290805, pages 1-12.

The agent that inhibits cyclic nucleotide phosphodiesterases can beselected from, tadalafil, vardenafil, udenafil, sildenafil and avanafil.

Treatment of Autoimmune, Autoinflammatory, Inflammatory, andCardiovascular Diseases

A therapeutic agent that binds to the CD47 protein for the treatment ofan autoimmune, autoinflammatory, inflammatory disease and/orcardiovascular disease can be combined with one or more therapeuticagent(s) such as an antibody, a chemical small molecule, or biologic ora medical or surgical procedure which include, but are not limited tothe following. For the treatment of autoimmune, autoinflammatory andinflammatory diseases, the combined therapeutic agents are:hydroxychloroquine, leflunomide, methotrexate, minocycline,sulfasalazine, abatacept, rituximab, tocilizumab, anti-TNF inhibitors orblockers (adalimumab, etanercept, infliximab, certolizumab pegol,golimumab), non-steroidal anti-inflammatory drugs, glucocorticoids,corticosteroids, intravenous immunoglobulin, anakinra, canakinumab,rilonacept, cyclophosphamide, mycophenolate mofetil, azathioprine,6-mercaptopurine, belimumab, beta interferons, glatiramer acetate,dimethyl fumarate, fingolimod, teriflunomide, natalizumab,5-aminosalicylic acid, mesalamine, cyclosporine, tacrolimus,pimecrolimus, vedolizumab, ustekinumab, secukinumab, ixekizumab,apremilast, budesonide and tofacitinib. For the treatment ofatherosclerosis, the combined therapeutic agents or procedures are:medical procedures and/or surgery, including percutaneous coronaryintervention (coronary angioplasty and stenting), coronary artery bypassgrafting, and carotid endarterectomy; therapeutic agents, includingangiotensin-converting enzyme (ACE) inhibitors (including ramipril,quinapril, captopril, and enalapril), calcium channel blockers(including amiodipine, nifedipine, verapamil, felodipine and diltiazem),angiotensin-receptor blockers (including eposartan, olmesarten,azilsartan, valsartan, telmisartan, losartan, candesartan, andirbesartan), the combination of ezetimibe and simvastatin, PCSK9inhibitors (including alirocumab and evolocumab), anacetrapib, andHMG-CoA inhibitors (including atorvastatin, pravastatin, simvastatin,rosuvastatin, pitavastatin, lovastatin and fluvastatin). For thetreatment of heart failure, the combined therapeutic agents are: ACEinhibitors, angiotensin receptor blockers, angiotensin receptorneprilsyn inhibitors (including the combination of sacubitril andvalsartan), diuretics, digoxin, inotropes, beta blockers and aldosteroneantagonists. For the treatment of pumonary hypertension the combinedtherapeutic agents are: sildenafil, tadalafil, ambrisentan, bosentan,macitentan, riociguat, treprostinil, epoprostenol, iloprost, andselexipag.

As disclosed herein, the anti-CD47 mAb is administered before, at thesame time or after the combined therapeutic agents or medical orsurgical procedures.

Another useful class of compounds for the combination therapiescontemplated herein includes modulators of SIRPα/CD47 binding such asantibodies to SIRPα, as well as soluble protein fragments of thisligand, or CD47 itself, inhibiting binding of, or interfering withbinding of, SIRPα to CD47. It should be noted that the therapeuticmethods encompassed herein include the use of the antibodies disclosedherein alone, in combination with one another, and/or withantigen-binding fragments thereof as well, for example, all possiblecombinations of these antibody compounds.

The examples illustrate various embodiments of the present disclosure,but should not be considered as limiting the disclosure to only theseparticularly disclosed embodiments.

Diagnostics for CD47 Expression

Diagnostics (including complementary and companion) have been an area offocus in the field of oncology. A number of diagnostic assays have beendeveloped for targeted therapeutics such as Herceptin (Genentech),Tarceva (OSI Pharmaceuticals/Genentech), Iressa (Astra Zeneca), andErbitux (Imclone/Bristol Myers Squibb). The anti-CD47 mAbs antibodies ofthe disclosure are particularly well-suited to use in diagnosticapplications. Accordingly, the disclosure provides a method to measureCD47 expression in tumor and/or immune cells, using an anti-CD47 mAb ofthe disclosure.

The anti-CD47 mAbs of the disclosure may be used in a diagnostic assayand/or in vitro method to measure CD47 expression in tumor and/or immunecells present in a patient's tumor sample. In particular, the anti-CD47mAbs of the disclosure may bind CD47 on approximately 1% or more oftumor and/or immune cells present in a patient's sample as compared to areference level. In another embodiment, the anti-CD47 mAbs may bind CD47on approximately 5% or more of tumor and/or immune cells in a patient'ssample as compared to a reference level, for example, or binding atleast 10%, or at least 20%, or at least 30%, or at least about 40%, orat least about 50%, or at least about 60%, or at least about 70%, or atleast about 80%, or at least about 90%, or between 10-100% as comparedto a reference level. In yet another embodiment, the anti-CD47 mAbs maybind CD47 on tumor and/or immune cells in a patient's sample to at leastabout a 2-fold increase as compared to a reference level, or at leastabout 3-fold, or at least about a 4-fold, or at least about a 5-fold orat least about a 8-fold increase, or between 2-fold and 8-fold, or about10-fold or greater as compared to a reference level. As describedherein, the measurement of CD47 expression in a patient's sampleprovides biological and/or clinical information that enables decisionmaking about the development and use of a potential drug therapy,notably the use of anti-CD47 antibodies for treating solid andhematological cancers, autoimmune disease, inflammatory disease,atherosclerosis, heart failure, in which the CD47 receptor plays a role.

In one embodiment, the in vitro method comprises, obtaining a patientsample, contacting the patient sample with a monoclonal antibody, orantigen-binding fragment thereof, which specifically binds to an epitopewithin the sequence of SEQ ID NO:66, and assaying for binding of theantibody to the patient sample, wherein binding of the antibody to thepatient sample is diagnostic of CD47 expression in a patient sample.

Accordingly, a diagnostic assay in accordance with the disclosure maycomprise contacting tumor and/or immune cells in a patient's sample withan anti-CD47 mAb, or an antigen binding fragment thereof, and assayingfor binding of the anti-CD47 mAb to a patient's tumor sample, whereinbinding of the anti-CD47 mAb to the patient sample is diagnostic of CD47expression. Preferably, the patient's sample is a sample containingtumor cells. In this case, binding of the anti-CD47 mAb of thedisclosure, or antigen binding fragment thereof, to the tumor cells maybe assessed for CD47 expression. The levels of CD47 expression by tumorcells and/or immune cells of a patient's tumor sample may be predictiveof clinical outcome in a patient.

Increased binding of anti-CD47 mAbs binding to cells in a patient'ssample is associated with increased CD47 expression. In one embodiment,the anti-CD47 mAbs of the disclosure may bind to approximately 5% ormore of tumor cells in a patient's sample and this may indicate that thepatient would benefit from rapid intervention to a solid andhematological cancer. A diagnostic assay of this sort may be used todetermine suitable therapeutic regimes for solid and hematologicalcancers with relatively high binding of anti-CD47 mAbs of thedisclosure, i.e., increased CD47 expression.

It will be appreciated that the diagnostic assay disclosed herein has anumber of advantages. The most important of these advantages is that thediagnostic assay of the disclosure may allow the user a greater deal ofconfidence in the CD47 expression in tumor and/or immune cells. Theincreased sensitivity of the diagnostic assay of the disclosure allowsdetection of CD47 in a patient's sample at lower levels than haspreviously been the case.

The anti-CD47 mAbs of the disclosure may be used as a diagnostic assayin relation to many forms of cancer. Particular forms of cancer that mayadvantageously be investigated for CD47 expression include susceptiblehematologic cancers and solid tumors including, but not limited to,leukemias, lymphomas, and solid tumors.

The diagnostic assays of the disclosure may utilize any suitable meansfor detecting binding of an anti-CD47 mAb to measure CD47 expression.Suitable methods may be selected with reference to the nature of anyreporter moiety used to label the anti-CD47 mAbs of the disclosure.Suitable techniques include, but are by no means limited to, flowcytometry, and enzyme linked immunosorbent assays (ELISA) and assaysutilizing nanoparticles.

EXAMPLES Example 1 Amino Acid Sequences Light Chain CDRs

LCDR1 LCDR2 LCDR3 Vx4-LCDR1 Vx4-LCDR2 Vx4-LCDR3 RSRQSIVHTNGNTYLG KVSNRFSFQGSHVPYT (SEQ ID NO: 11) (SEQ ID NO: 15) (SEQ ID NO: 18) Vx8-LCDR1Vx8-LCDR2 Vx8-LCDR3 RASQDISNYLN YTSRLYS QQGNTLPWT (SEQ ID NO: 12)(SEQ ID NO: 16) (SEQ ID NO: 19) Vx8-LCDR1 RASQSISNYLN (SEQ ID NO: 13)Vx9-LCDR1 Vx9-LCDR2 Vx9-LCDR3 RSSQNIVQSNGNTYLE KVFHRFS FQGSHVPWT(SEQ ID NO: 14) (SEQ ID NO: 17) (SEQ ID NO: 20)

Heavy Chain CDRs

HCDR1 HCDR2 HCDR3 Vx4-HCDR1 Vx4-HCDR2 Vx4-HCDR3 GYTFTNYVIHYIYPYNDGILYNEKFKG GGYYVPDY (SEQ ID NO: 1) (SEQ ID NO: 4) (SEQ ID NO: 7)Vx4-HCDR3 GGYYVYDY (SEQ ID NO: 8) Vx8-HCDR1 Vx8-HCDR2 Vx8-HCDR3GYSFTNYYIH YIDPLNGDTTYNQKFKG GGKRAMDY (SEQ ID NO: 2) (SEQ ID NO: 5)(SEQ ID NO: 9) Vx9-HCDR1 Vx9-HCDR2 Vx9-HCDR3 GYTFTNYWIHYTDPRTDYTEYNQKFKD GGRVGLGY (SEQ ID NO: 3) (SEQ ID NO: 6) (SEQ ID NO: 10)

Murine Light Chain Variable Domains

>Vx4murL01 (SEQ ID NO: 41)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIK. >Vx4murL02 (SEQ ID NO: 42)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIK. >Vx8murL03 (SEQ ID NO: 46)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGG GTKLEIK. >Vx9murL04(SEQ ID NO: 50) DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVP WTFGGGTKVEIK 

Murine Heavy Chain Variable Domains

>Vx4murH01 (SEQ ID NO: 21)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSS. >Vx4mur-H02 (SEQ ID NO: 22)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTLVTVSS. >Vx8murH03 (SEQ ID NO: 28)EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSVTVSS. >Vx9murH04 (SEQ ID NO: 35)QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGG RVGLGYWGHGSSVTVSS

Human Light Chain Variable Domains

>Vx4humL01 (SEQ ID NO: 43)DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIK >Vx4humL02 (SEQ ID NO: 44)DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK >Vx4humL03 (SEQ ID NO: 45)DIVMTQSPDSLAVSLGERATINCRSRQSIVHTNGNTYLGWYQQKPGQPPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIK >Vx8humL04 (SEQ ID NO: 47)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTF GQGTKVEIK. >Vx8humL05(SEQ ID NO: 48) DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTF GQGTKVEIK. >Vx8humL06(SEQ ID NO: 49) DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTF GQGTKLEIK >Vx9humL07(SEQ ID NO: 51) DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIK. >Vx9humL08 (SEQ ID NO: 52)DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVP YTFGQGTKLEIK.

Human Heavy Chain Variable Domains

>Vx4humH01 (SEQ ID NO: 23)QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATLVTVSS. >Vx4humH02 (SEQ ID NO: 24)QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATLVTVSS. >Vx4humH03 (SEQ ID NO: 25)EVQLVQSGAEVKKPGATVKISCKVSGYTFTNYVIHWVQQAPGKGLEWMGYIYPYNDGILYNEKFKGRVTITADTSTDTAYMELSSLRSEDTAVYYCATGGYYVPDYWGQGTTVTVSS >Vx4humH04 (SEQ ID NO: 26)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTTVTVSS >Vx4humH05 (SEQ ID NO: 27)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGTTVTVSS >Vx8humH06 (SEQ ID NO: 29)QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH07 (SEQ ID NO: 30)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH08 (SEQ ID NO: 31)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH09 (SEQ ID NO: 32)QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSS. >Vx8humH10 (SEQ ID NO: 33)EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTLVTVSS. >Vx8humH11 (SEQ ID NO: 34)QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQATLVTVSS >Vx9humH12 (SEQ ID NO: 36)QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH13 (SEQ ID NO: 37)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH14 (SEQ ID NO: 38)[1]EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH15 (SEQ ID NO: 39)QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSS. >Vx9humH16 (SEQ ID NO: 40)EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGG RVGLGYWGQGTLVTVSS.

Human IgG-Fc

>Human Fc IgG1 (SEQ ID NO: 53)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc IgG1-N297Q (SEQ ID NO: 54)ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc-IgG2 (SEQ ID NO: 56)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Human Fc-IgG3 (SEQ ID NO: 57)ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHEALHNRFTQKSLSLSPGK >Human Fc-IgG4 (SEQ ID NO: 58)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. >Human Fc-IgG4 S228P (SEQ ID NO: 59)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG. >Human Fc-IgG4PE (SEQ ID NO: 60)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK >Human Fc-IgG4PE′ (SEQ ID NO: 101)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG >Human kappa LC (SEQ ID NO: 61)RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Rat Fc-IgG2c (SEQ ID NO: 62)ARTTAPSVYPLVPGCSGTSGSLVTLGCLVKGYFPEPVTVKWNSGALSSGVHTFPAVLQSGLYTLSSSVTVPSSTWSSQTVTCSVAHPATKSNLIKRIEPRRPKPRPPTDICSCDDNLGRPSVFIFPPKPKDILMITLTPKVTCVVVDVSEEEPDVQFSWFVDNVRVFTAQTQPHEEQLNGTFRVVSTLHIQHQDWMSGKEFKCKVNNKDLPSPIEKTISKPRGKARTPQVYTIPPPREQMSKNKVSLTCMVTSFYPASISVEWERNGELEQDYKNTLPVLDSDESYFLYSKLSVDTDSWMRGDIYTCSVVHEALHNHHTQKNLSRSPGK. >Rat kappa LC (SEQ ID NO: 63)RADAAPTVSIFPPSMEQLTSGGATVVCFVNNFYPRDISVKWKIDGSEQRDGVLDSVTDQDSKDSTYSMSSTLSLTKVEYERHNLYTCEVVHKTSSSPVVK SFNRNEC.

Rabbit IgG-Fc

>Rabbit IgG (SEQ ID NO: 64)GQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK. >Rabbit kappa LC (SEQ ID NO: 65)RDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFN RGDC. >CD47(SEQ ID NO: 66) MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVKWKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCEVTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTIALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIAILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVE.

Chimera and Human Light Chains

>Vx4murL01 Full length (SEQ ID NO: 67)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx4murL01 Full length (SEQ ID NO: 68)DVLMTQTPLSLPVNLGDQASISCRSRQSIVHTNGNTYLGWFLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLTISRVEAEDLGVYYCFQGSHVPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx4humL01 Full length LC (SEQ ID NO: 69)DIVMTQSPLSLPVTPGEPASISCRSRQSIVHTNGNTYLGWYLQKPGQSPRLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8humL03 Full length LC (SEQ ID NO: 70)DIVMTQSPLSLPVTPGEPASISCRASQDISNYLNWYLQKPGQSPRLLIYYTSRLYSGVPDRFSGSGSGTDFTLKISRVEADDVGIYYCQQGNTLPWTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx9humL02 Full length LC (SEQ ID NO: 71)DIVMTQSPDSLAVSLGERATINCRSSQNIVQSNGNTYLEWYQQKPGQPPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8humL02 Full length LC (SEQ ID NO: 72)DIQMTQSPSSLSASVGDRVTITCRASQSISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx4humL02 Full length LC (SEQ ID NO: 73)DVVMTQSPLSLPVTLGQPASISCRSRQSIVHTNGNTYLGWFQQRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx9humL07 Full length LC (SEQ ID NO: 74)DVVMTQSPLSLPVTLGQPASISCRSSQNIVQSNGNTYLEWFQQRPGQSPRRLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8humL01 Full length LC (SEQ ID NO: 75)DIQMTQSPSSLSASVGDRVTITCRASQDISNYLNWYQQKPGKAPKLLIYYTSRLYSGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGNTLPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx8murL03 Full length LC (SEQ ID NO: 76)DIQMTQTTSSLSASLGDRVTISCRASQDISNYLNWYQQKPDGTVKLLIYYTSRLYSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPWTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC. >Vx9mur_L04 Full length LC (SEQ ID NO: 77)DVFMTQTPLSLPVSLGDQASISCRSSQNIVQSNGNTYLEWYLQKPGQSPKLLIYKVFHRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPWTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGEC.

Chimera and Human Heavy Chains

>Vx4murH01 Full length HC  (SEQ ID NO: 78)EVQLQQSGPELVKPGASVKMSCKASGYTFTNYVIHWVKRRPGQGLEWIGYIYPYNDGILYNEKFKGKATVTSDKSSSTAYMDLSSLTSEDSAVYYCTRGGYYVPDYWGQGTTLTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH01 Full length HC (SEQ ID NO: 79) QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVPDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH11 Full length HC (SEQ ID NO: 80) QVQLVQSGAEVKKPGASVQVSCKASGYSFTNYYIHWLRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGKRAMDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH12 Full length HC (SEQ ID NO: 81) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx9humH14 Full length HC (SEQ ID NO: 82) EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYNQKFKDQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx9humH15 Full length HC (SEQ ID NO: 83) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx4humH02 Full length HC (SEQ ID NO: 84) QVQLVQSGAEVKKPGASVQVSCKASGYTFTNYVIHWLRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTSDTSISTAYMELSSLRSDDTAVYYCARGGYYVYDYWGQATLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH13 Full length HC (SEQ ID NO: 85) QVQLVQSGAEVKKPGSSVKVSCKASGYTFTNYWIHWVRQAPGQGLEWMGYTDPRTDYTEYNQKFKDRVTITADESTSTAYMELSSLRSEDTAVYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH10 Full length HC (SEQ ID NO: 86) EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGRGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH04 Full length HC (SEQ ID NO: 87) EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYVIHWVRQMPGKGLEWMGYIYPYNDGILYNEKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGYYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx4humH05 Full length HC (SEQ ID NO: 88) QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYVIHWVRQAPGQGLEWMGYIYPYNDGILYNEKFKGRVTMTTDTSTSTAYMELRSLRSDDTAVYYCARGGYYVPDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9humH16 Full length HC (SEQ ID NO: 89) EVQLVQSGAEVKKPGESLKISCKGSGYTFTNYWIHWVRQMPGKGLEWMGYTDPRTDYTEYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGRVGLGYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH06 Full length HC (SEQ ID NO: 90) [2]QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH07 Full length HC (SEQ ID NO: 91) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH08 Full length HC (SEQ ID NO: 92) EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH09 Full length HC (SEQ ID NO: 93) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8humH06 Full length HC (SEQ ID NO: 94) QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx8mur-H03 Full length HC (SEQ ID NO: 95) EVQLQQSGPELMKPGASVKISCKASGYSFTNYYIHWVNQSHGKSLEWIGYIDPLNGDTTYNQKFKGKATLTVDKSSSTAYMRLSSLTSADSAVYYCARGGKRAMDYWGQGTSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK. >Vx9mur-H04 Full length HC (SEQ ID NO: 96) QVQLQQFGAELAKPGASVQMSCKASGYTFTNYWIHWVKQRPGQGLEWIGYTDPRTDYTEYNQKFKDKATLAADRSSSTAYMRLSSLTSEDSAVYYCAGGGRVGLGYWGHGSSVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH06 Full length HC (SEQ ID NO: 97) QVQLVQSGAEVKKPGASVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH07 Full length HC (SEQ ID NO: 98) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYNQKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH08 Full length HC (SEQ ID NO: 99) EVQLVQSGAEVKKPGESLKISCKGSGYSFTNYYIHWVRQMPGKGLEWMGYIDPLNGDTTYNQKFKGQVTISADKSISTAYLQWSSLKASDTAMYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK. >Vx8humH09 Full length HC (SEQ ID NO: 100) QVQLVQSGAEVKKPGSSVKVSCKASGYSFTNYYIHWVRQAPGQGLEWMGYIDPLNGDTTYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARGGKRAMDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

Example 2 Production of CD47 Antibodies

Chimeric antibodies disclosed herein comprise a mouse heavy chainvariable domain and a light chain variable domain combined with a humankappa or human Fc IgG1, IgG1-N297Q, IgG2, IgG4, IgG4 S228P, and IgG4 PEconstant domains, respectively. These were designed to incorporate asecretion signal and cloned into a mammalian expression system, andtransfected into CHO cells to generate chimeric (murine-human)antibodies. The chimeric variants were expressed as full length IgGmolecules, secreted into the medium, and purified using protein A.

Multiple methods for humanizing antibodies are well-known to those ofordinary skill in the art. One such method, as used herein, haspreviously been described (Making and Using Antibodies a PracticalHandbook, Second Edition, Ed. Matthew R. Kase, Chapter 15: Humanizationof Antibodies, Juan Carlos Almagro et al., CRC Press 2013). As such, thehumanized antibodies disclosed herein comprise frameworks derived fromthe human genome. The collection covers the diversity found in the humangerm line sequences, yielding functionally expressed antibodies in vivo.The complementarity determining regions (CDRs) in the light and heavychain variable regions of the murine and chimeric (murine-human) aredescribed herein and were determined by following commonly acceptedrules disclosed in “Protein Sequence and Structure Analysis of AntibodyVariable Domains,” In: Antibody Engineering Lab Manual, eds. S. Duebeland R. Kontermann, Springer-Verlag, Heidelberg (2001)). The human lightchain variable domains were then designed. The humanized variabledomains were then combined with a secretion signal and human kappa andhuman Fc IgG1, IgG1-N297Q, IgG2, IgG3, IgG4 S228P and IgG4 PE constantdomains, cloned into a mammalian expression system, and transfected intoCHO cells to generate humanized mAbs. The humanized variants wereexpressed as full length IgG molecules, secreted into the medium andpurified using protein A.

A non-glycosylated version (IgG1-N297Q) was created by site directedmutagenesis of heavy chain position 297 to change the asparagine toglutamine (Human Fc IgG1-N297Q, SEQ ID NO:54). An IgG4 variant wascreated by site-directed mutagenesis at position 228 to change theserine to proline thereby preventing in vivo Fab arm exchange. An IgG4double mutant was created by site-directed mutagenesis at positions 228(serine to proline) and 235 (leucine to glutamate) to prevent Fab armexchange and to further reduce Fc effector function. IgG2, IgG3, IgG4S228P, and IgG4PE isotypes were constructed by cloning the heavy chainvariable domain in frame with the human IgG2, IgG3, IgG4 S228P, andIgG4PE constant domains (Human Fc-IgG2, SEQ ID NO:56 Human Fc-IgG3, SEQID NO:57; Human Fc-IgG4 S228P, SEQ ID NO:59; and Human Fc-IgG4PE, SEQ IDNO:60).

Example 3 Binding of CD47 Monoclonal Antibodies (mAbs)

The binding of chimeric (murine-human) and humanized antibodies of thepresent disclosure was determined by ELISA using OV10 cells transfectedwith human CD47 (OV10 hCD47) or using freshly isolated human red bloodcells (hRBCs), which display CD47 on their surface (Kamel et al. (2010)Blood. Transfus. 8(4):260-266).

Binding activities of VLX4, VLX8, and VLX9 chimeric (xi) and humanizedmAbs were determined using a cell-based ELISA assay with human OV10hCD47 cells expressing cell surface human CD47. OV10 hCD47 cells weregrown in IMDM medium containing 10% heat inactivated fetal bovine serum(BioWest; S01520). One day before assay, 3×10⁴ cells were plated in 96well cell bind plates (Corning #3300, VWR #66025-626) and were 95-100%confluent at the time of assay. Cells were washed, variousconcentrations of purified antibodies added in IMDM and incubated at 37°C. for 1 hr in 95% O₂/5% CO₂. Cells were then washed with media andincubated for an additional hour at 37° C. with HRP labelled secondaryanti-human antibody (Promega) diluted 1/2500 in media. Cells were washedthree times with PBS, and the peroxidase substrate 3,3′,5,5′-tetramethylbenzidine was added (Sigma; Catalog #T4444). Reactionswere terminated by the addition of HCl to 0.7N, and absorbance at 450 nMdetermined using a Tecan model Infinite M200 plate reader. The apparentbinding affinities of these clones to human OV10 hCD47 cells wasdetermined by non-linear fit (Prism GraphPad software).

Binding activities of chimeric and humanized VLX4, VLX8, and VLX9 mAbsto human CD47 on hRBCs were also determined using flow cytometry. Bloodwas obtained from normal volunteers and RBCs were washed 3 times withphosphate buffered saline, pH 7.2 containing 2.5 mM EDTA (PBS+E). hRBCswere incubated for 60 min at 37° C. with various concentrations of thechimeric or humanized antibodies in a PBS+E. Cells were then washed withcold PBS+E and incubated for an additional hour on ice with FITClabelled donkey anti-human antibody (Jackson Immuno Research Labs, WestGrove, Pa.; Catalogue #709-096-149) in PBS+E. Cells were washed withPBS+E, antibody binding was analyzed using a C6 Accuri Flow Cytometer(Becton Dickinson) and apparent binding affinities determined bynon-linear fit (Prism GraphPad software) of the median fluorescenceintensities at the various antibody concentrations.

All of the VLX4 chimeric (murine-human) mAbs bound to human OV10 hCD47tumor cells with apparent affinities in the picomolar (pM) range (Table1).

Similarly, the humanized VLX4 mAbs bound to human OV10 hCD47 tumor cellsin a concentration-dependent manner (FIG. 1A and FIG. 1B) with apparentbinding affinities ranging from the picomolar to low nanomolar range(Table 2).

All of the chimeric VLX4 mAbs bound to human RBCs with apparent Kdvalues in the picomolar range and these were similar to the K_(d) valuesobtained for OV10 hCD47 tumor cells by ELISA (Table 1).

The humanized VLX4 mAbs VLX4hum_01 IgG1 N297Q, VLX4hum_02 IgG1 N297Q,VLX4hum_01 IgG4PE, VLX4hum_02 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13IgG4PE bound to human RBCs with Kd values similar to those obtained forOV10 hCD47 tumor cells whereas VLX4hum_06 IgG4PE and VLX4hum_07 IgG4 PEexhibited reduced binding to hRBCs (FIG. 2A, FIG. 2B, and Table 2). Thisdifferential binding of the humanized mAbs to tumor cells and RBCs wasunexpected as the VLX4 IgG4PE chimeric mAb bound with similar apparentKd values to both tumor and RBC CD47 (Table 1).

As shown in Table 1, all the VLX8 chimeric mAbs bound to human OV10hCD47 tumor cells in a concentration-dependent manner with apparentaffinities in the picomolar (pM) range.

Similarly, the humanized VLX8 mAbs bound to human OV10 hCD47 tumor cellsin a concentration-dependent manner (FIG. 3A and FIG. 3B) with apparentaffinities in the picomolar range (Table 2).

All the VLX8 chimeric mAbs bound to hRBCs with apparent K_(d) values inthe picomolar range and these were similar to the apparent K_(d) valuesobtained for OV10 hCD47 tumor cells by ELISA (Table 1).

The VLX8 humanized mAbs VLX8hum_01 IgG4PE, VLX8hum_02 IgG4PE, VLX8hum_03IgG4PE, VLX8hum_04 IgG4PE, VLX8hum_05 IgG4 PE, and VLX8hum_06 IgG4PE,VLX8hum_07 IgG4PE, VLX8hum_08 IgG4 PE, VLX8hum_09 IgG4 PE, VLX8hum_11IgG4 PE, VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 and VLX8hum_09IgG2 IgG2 bound to human RBCs with Kd values similar to the valuesobtained for OV10 hCD47 tumor cells whereas VLX8hum_10 IgG4PE exhibitedreduced to hRBCs (FIG. 4A, FIG. 4B, and Table 2). This differentialbinding of the humanized mAbs to tumor cells and RBCs was unexpected asthe VLX8 IgG4PE chimeric mAb bound with similar apparent Kd values toboth tumor and RBC CD47 (Table 1).

Table 1 shows the apparent binding affinities of VLX9 chimeric mAbs tohuman OV10 hCD47 cells and to human RBCs. All of the chimeric mAbs boundto OV10 hCD47 tumor cells with apparent binding constants in thepicomolar range. Similarly, the humanized VLX9 mAbs bound to human OV10hCD47 tumor cells in a concentration-dependent manner (FIG. 5A and FIG.5B) with apparent affinities in the picomolar to nanomolar range (Table2).

All the VLX9 chimeric mAbs bound to hRBCs with apparent Kd values in thepicomolar range and these were similar to the apparent K_(d) valuesobtained for OV10 hCD47 tumor cells by ELISA (Table 1). In contrast tothe chimeric mAbs, the VLX9 humanized mAbs VLX9hum_01 IgG2, VLX9hum_02IgG2 and VLX9hum_07 IgG2 exhibited reduced binding to human RBCs (FIG.7, Table 2). By contrast, the humanized mAbs VLX9hum_03 IgG2, VLX9hum_04IgG2, VLX9hum_05 IgG2, VLX9hum_06 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2and VLX9hum_10 IgG2 exhibited no measureable binding to RBCs up to 5,000pM (Table 2). This differential binding of the humanized mAbs to tumorcells and RBCs was unexpected as the VLX9 IgG2 chimeric mAbs all boundwith similar apparent Kd values to both tumor and RBC CD47 (Table 1).

Specific binding of CD47 humanized mAbs was demonstrated using Jurkatwildtype and Jurkat CD47 knockout (KO) cells. Jurkat wildtype and JurkatCD47 KO cells were grown in RPMI medium containing 10% heat inactivatedfetal bovine serum (BioWest; S01520). The cells were washed and 1×10⁴cells were resuspended media and incubated with various antibodyconcentrations for one hour at 370 in 5% CO₂. Cells were then washedtwice with 1×PBS and then resuspended 1:1000 in secondary antibody (goatanti-human IgG (H+L) FITC-labelled, Jackson Labs, 109-095-003) for onehour at 37° in 5% CO₂. Cells were then washed twice with 1×PBS andresuspended in 1×PBS. Median fluorescence intensity was determined byflow cytometry and the apparent binding affinities determined bynon-linear fit (Prism GraphPad software).

As shown in FIG. 6, VLX4hum_07 IgG4PE (FIG. 6A) and VLX9hum_09 IgG2(FIG. 6B) bound to Jurkat cells expressing CD47, whereas no binding isobserved to Jurkat CD47KO cells.

TABLE 1 Binding of VLX4, VLX8, and VLX9 Chimeric (xi) mAbs to OV10 hCD47Cells and Human Red Blood Cells (hRBCs). Kd (pM) OV10 hCD47 Kd (pM) HACell-based ELISA hRBC hRBC VLX4 IgG1 (xi) 315 104 Yes VLX4 IgG1 N297Q(xi) 258 92 Yes VLX4 IgG2 (xi) 431 184 Yes VLX4 IgG4 S228P (xi) 214 99No VLX4 IgG4 PE(xi) 225 303 No VLX8 IgG1 N297Q (xi) 42 91 Yes VLX8 IgG4PE (xi) 56 77 Yes VLX9 IgG1 (xi) 280 381 Yes VLX9 IgG1 N297Q (xi) 275190 Yes VLX9 IgG2 (xi) 880 742 Yes VLX9 IgG4 PE (xi) 293 126 Yes

TABLE 2 Binding of VLX4, VLX8, and VLX9 Humanized mAbs to Human OV10hCD47 and Human Red Blood Cells (hRBCs). Kd (pM) OV10 hCD47 Kd (pM) HACell-based ELISA hRBC hRBC VLX4hum_01 IgG1 73  23 Yes VLX4hum_02 IgG1 80 70 Yes VLX4hum_01 IgG4 PE 82  80 No VLX4hum_02 IgG4 PE 95  75 R***VLX4hum_06 IgG4 PE 196 >33,000**   Yes VLX4hum_07 IgG4 PE209 >33,000**   Yes VLX4hum_12 IgG4 PE 56 263 Yes VLX4hum_13 IgG4 PE 62340 Yes VLX8hum_01 IgG4 PE 54 209 No VLX8hum_02 IgG4 PE 50 221 NoVLX8hum_03 IgG4 PE 67 183 No VLX8hum_04 IgG4 PE 49 119 No VLX8hum_05IgG4 PE 68 264 No VLX8hum_06 IgG4 PE 61 274 Yes VLX8hum_07 IgG4 PE 24241 Yes VLX8hum_08 IgG4 PE 97 217 Yes VLX8hum_09 IgG4 PE 82 336 YesVLX8hum_10 IgG4 PE 183 >33,000**   Yes VLX8hum_11 IgG4 PE 90  87 NoVLX8hum_06 IgG2 403 246 Yes VLX8hum_07 IgG2 460 671 Yes VLX8hum_08 IgG2464 820 Yes VLX8hum_09 IgG2 680 1739  Yes VLX9hum_01 IgG2 162  1653** NoVLX9hum_02 IgG2 227  4103** No VLX9hum_03 IgG2 606 *MB No VLX9hum_04IgG2 823 *MB No VLX9hum_05 IgG2 6372 *MB No VLX9hum_06 IgG2 547 *MB NoVLX9hum_07 IgG2 341 >66,000**   ***R VLX9hum_08 IgG2 688 *MB NoVLX9hum_09 IgG2 8340 *MB No VLX9hum_10 IgG2 12232 *MB No *MB—Minimalbiniding; no measurable binding detected at mAb concentration up to5,000 pM. **Reduced RBC binding. ***R—Reduced hemagglutination.

Cross-species binding of humanized VLX4, VLX8, and VLX9 mAbs wasdetermined using flow cytometry. Mouse, rat, rabbit or cynomolgus monkeyRBCs were incubated for 60 min on at 37° C. with various concentrationsof the humanized antibodies in a solution of phosphate buffered saline,pH 7.2, 2.5 mM EDTA (PBS+E). Cells were then washed with cold PBS+E, andincubated for an additional hr on ice with FITC labelled donkeyanti-human antibody (Jackson Immuno Research Labs, West Grove, Pa.;Catalogue #709-096-149) in PBS+E. Cells were washed with PBS+E, andantibody binding analyzed using a C6 Accuri Flow Cytometer (BectonDickinson).

Table 3 shows the apparent binding affinities of the humanized VLX4 andVLX8 mAbs to RBCs from mouse, rat, and cynomolgus monkey determined bynon-linear fit (Prism GraphPad software) of the median fluorescenceintensities at various antibody concentrations. This data demonstratesthat humanized VLX4 and VLX8 mAbs bind to mouse, rat, rabbit (data notshown) and cynomolgus monkey RBCs with apparent Kd values in thepicomolar to nanomolar range.

TABLE 3 Binding of VLX4 and VLX8 Humanized mAbs to Mouse, Rat andCynomolgus Monkey RBCs. Kd (pM) Kd (pM) Kd (pM) Cynomolgus Mouse RBC RatRBC Monkey RBC VLX4hum_01 IgG4 PE 13001 30781 56 VLX4hum_07 IgG4 PE15192 14274 13522 VLX8hum_11 IgG4 PE 9123 8174 55

Example 4 Binding of Humanized Anti-CD47 mAbs Determined by SurfacePlasmon Resonance

Binding of soluble anti-CD47 mAbs to recombinant human His-CD47 wasmeasured in vitro by surface plasmon resonance on a Biacore 2000. AnAnti-Human IgG (GE Lifesciences) was amine coupled to a CM5 chip on flowcells 1 and 2. The humanized mAbs VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_08 IgG2 or VLX9hum_03 IgG2 diluted in HBS-EP⁺ running buffer (pH7.2) were captured onto flow cell 2. Multi-cycle kinetics weredetermined using 0 to 1000 nM His-tagged human CD47 (Acro Biosystems)diluted in HBS-EP⁺ running buffer (pH 7.2) with contact time of 180seconds and dissociation time of 300 seconds. A 1:1 binding model wasemployed for kinetic analysis of binding curves. The on-rate, off-rateand Dissociation constants for VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_08 IgG2 and VLX9hum_03 IgG2 are shown in Table 4.

TABLE 4 Binding of VLX4, VLX8 and VLX9 Humanized mAbs to HumanRecombinant His-CD47 by Surface Plasmon Resonance at pH 7.2. k_(a) k_(d)K_(D) (nM) VLX4hum_07 IgG4PE 1.7e⁵ 8.7e⁻⁴ 5.1 VLX8hum_11 IgG4PE 6.8e⁵7.9e⁻⁴ 1.2 VLX9_08 IgG2 7.6e⁴ 6.5e⁻⁴ 8.6 VLX9_03 IgG2 6.5e⁴ 7.3e⁻⁴ 11.1

Example 5 Differential Binding of Anti-CD47 mAbs

Some soluble CD47 antibodies described herein have been shown todifferentially bind to normal cells. This additional property ofselective binding is expected to have advantages compared to mAbs thatbind with equal affinity to normal and tumor cells. Anti-CD47 mAbs withsuch reduced binding have not been described.

Binding by soluble anti-CD47 mAbs is measured in vitro. Bindingactivities of VLX4, VLX8, and VLX9 humanized mAbs were determined usinga flow cytometry based binding assay with human aortic endothelial cells(HAEC), skeletal muscle cells (SkMC), human lung microvascularendothelial cells (HMVEC-L), renal tubular epithelial cells (RTEC), CD3⁺cells or peripheral blood mononuclear cells (PBMC). HAEC, SkMC, HMVEC-Land RTEC cells were purchased from Lonza and cultured according to themanufacturer's recommendations. Adherent cells were removed from theculture flask with accutase, resuspended in the recommended media and1×10⁴ cells were incubated with various antibody concentrations for onehour at 37°, 5% CO₂. For non-adherent cells, 1×10⁴ cells wereresuspended in the recommended media and incubated with various antibodyconcentrations for one hour at 37°, 5% CO₂. Cells were then washed twicewith 1×PBS and then resuspended 1:1000 in secondary antibody (goatanti-human IgG (H+L)—FITC, Jackson Labs, 109-095-003) for one hour at37° C., 5% CO₂.

PBMC were isolated by ficoll gradient and were incubated with an FcRblocking reagent (Miltenyi Biotec) for 10 min at 4° C. permanufacturer's recommendation immediately preceeding the addition ofvarious concentrations of antibodies diluted in PBS. CD3 cells weredetected using an allophycocyanin (APC)-labelled anti-CD3 antibody (BDBioSciences) which was added at the same time as the FITC-labelled goatanti-human IgG (H+L) antibody. Cells were washed twice with 1×PBS andantibody binding was assessed by flow cytometry analysis.

As shown in FIG. 8A, VLX4 and VLX8 humanized mAbs bound to HAEC cellswhereas VLX9 humanized mAbs had reduced or minimal binding to HAEC cellsas compared to tumor cells (Table 5). VLX9 humanized mAbs also showedreduced binding to SkMC cells (FIG. 8B), reduced or minimal binding toHMVEC-L cells (FIG. 8C), reduced binding to RPTEC cells (FIG. 8D) ascompared to binding to tumor cells (Table 5). Reduced binding of VLX9humanized mAbs was also observed to CD3⁺ cells (FIG. 8E) and PBMC (FIG.8F) as compared to tumor cells (Table 5). This selective binding impartsan additional desirable antibody property and potential therapeuticbenefit in the treatment of cancer.

TABLE 5 VLX4, VLX8 and VLX9 Humanized mAbs Binding to Normal Cells. Kd(pM) OV10 hCD47 Cell-based Kd (pM) Kd (pM) Kd (pM) Kd (pM) Kd (pM) Kd(pM) Kd (pM) ELISA hRBC HAEC HMVEC-L SKMC RPTEC CD3⁺ PBMC VLX4hum_01IgG4 PE 82    80    118     72     5     26  220  269 VLX4hum_07 IgG4 PE209 >33,000**    747    792    630    784  440  499 VLX8hum_10 IgG4 PE183 >33,000**    1104    2113**    461    491   91  106 VLX8hum_11 IgG4PE 90    87     34     20     7     26  144  156 VLX9hum_03 IgG2 606 MB*MB* >200,000** >200,000** >200,000** 10863** 10232** VLX9hum_04 IgG2 823MB* MB* MB* >200,000** >200,000**  7426**  7619** VLX9hum_06 IgG2 547MB* >200,000**   71619**   23483**    4847** 19354** 17904** VLX9hum_08IgG2 688 MB* >200,000** >200,000**   34783** >200,000** 28287** 24486**VLX9hum_09 IgG2 8340 MB* MB* MB* MB* >200,000** 56146** 48348***MB—Minimal binding, no measureable binding detected at mAbconcentration up to 5,000 pM. **Reduced binding.

Example 6 pH Dependent and Independent Binding of Humanized Anti-CD47mAbs

Some soluble anti-CD47 mAbs described herein have been shown to bindtumor cells at acidic pH with greater affinity compared to physiologicpH. This additional property is expected to have advantages compared tomAbs that bind at similar affinities to CD47 at both acidic andphysiologic pH, in part due to the acidic nature of the tumormicroenvironment (Tannock and Rotin, Cancer Res 1989; Song et al. CancerDrug Discovery and Development 2006; Chen and Pagel, Advan Radiol 2015).

Binding by soluble anti-CD47 mAbs to immobilized recombinant human CD47and to human CD47 expressed on cells was measured in vitro. For the invitro binding to recombinant CD47, His-CD47 (AcroBiosystems) wasadsorbed to high-binding microtiter plates overnight at 4° C. The wellswere washed and varying concentrations of anti-CD47 mAbs were added tothe wells in buffers with a of either pH 6 or pH 8 for 1 hour. The wellswere washed and then incubated with HRP-labelled secondary antibody for1 hour at pH 6 or pH 8 followed by addition of peroxidase substrate. Theapparent affinities were calculated using non-linear fit model (GraphpadPrism).

For analysis of pH dependent binding by surface plasmon resonance usinga Biacore 2000, an Anti-Human IgG (GE Lifesciences) was amine coupled toa CM5 chip on flow cells 1 and 2. An Fc-tagged human CD47 (AcroBiosystems) was diluted in PBS-EP⁺ running buffer (pH 7.5, 6.5 or 6.0)and captured onto flow cell 2. Multi-cycle kinetics were determinedusing 0 to 100 nM VLX8hum_11 Fab or VLX9hum_08 Fab diluted in PBS-EP⁺running buffer (pH 7.5, 6.5 or 6.0) with contact time of 180 seconds anddissociation time of 300 seconds. A 1:1 binding model was employed forkinetic analysis of binding curves.

For the in vitro binding to cells expressing CD47, Jurkat cells weregrown in RPMI medium containing 10% heat inactivated fetal bovine serum(BioWest; S01520). The cells were washed and 1×10⁴ cells wereresuspended in PBS supplementated with 2% FBS at either pH 7.4 or 6.5and incubated with various antibody concentrations for 1 hour at 37° C.Cells were then washed twice and resuspended with 1:1000 of secondaryantibody (goat anti-human IgG (H+L) labelled with Alexa488,JacksonImmunoresearch) for 1 hour at 37° C. at pH 6 or pH 8. Cells werethen washed twice and the median fluorescence intensity was determinedby flow cytometry. The apparent binding affinities were determined bynon-linear fit (Prism GraphPad software).

As shown in FIG. 9A and FIG. 9B, the soluble VLX9 humanized mAbs(VLX9hum_09 IgG2 and VLX9hum_04 IgG2) bound to His-CD47 with greateraffinity at the more acidic pH 6.0 than at pH 8.0. Neither VLX4hum_07IgG4PE (FIG. 9C) nor VLX8hum_10 IgG4PE (FIG. 9D) displayed pH dependentbinding. In addition, the murine VLX9 antibody and VLX9 chimericantibodies containing human Fc from isoytpes IgG1, IgG2 and IgG4PE didnot display pH dependence (Table 6) whereas VLX9hum_04 as either an IgG,IgG2 or an IgG4PE demonstrated greater binding to His-CD47 at acidic pH(Table 7). The apparent binding affinities for additional humanized mAbsto recombinant human CD47 are shown in Table 8. All humanized VLX9 mAbsexhibited pH dependent binding whereas the VLX4 and VLX8 humanized mAbsdid not. To determine the effect of pH on on-rates, off-rates anddissociation constants, Biacore analysis was performed for humanizedmAbs VLX8hum_11 Fab fragment and VLX9hum_08 Fab at pH 6, pH 6.5 and pH7.5. The VLX9hum_08 Fab exhibited pH dependent binding that increasedwith decreasing pH wheras the VLX8hum_11 Fab did not. The on-rate,off-rate and dissociation constants for VLX8hum_11 Fab and VLX9hum_08Fab are shown in Table 9. Table 10 illustrates the pH dependent bindingexhibited by VLX9hum_04 IgG2 to CD47 expressed on Jurkat cells. No pHdependent binding was exhibited by VLX4hum_07 IgG4PE. This pH dependenceof the VLX9 humanized mAbs imparts an additional desirable antibodyproperty and therapeutic benefit in the treatment of cancer.

TABLE 6 Murine VLX9 and mouse-human chimeric VLX9 Binding to CD47 is notpH Dependent KD (pM) KD (pM) pH 6 pH 8 VLX9 IgG (murine) 91 76 VLX9IgG1-N297Q (xi) 99 135 VLX9 IgG2 (xi) 130 137 VLX9 IgG4PE (xi) 133 160

TABLE 7 VLX9hum_04 Humanized mAbs Bind to CD47 in a pH Dependent Mannerand Binding is not Isotype Specific KD (pM) KD (pM) pH 6 pH 8 VLX9hum_04Ig1-N297Q 215 >33,000 VLX9hum_04 IgG2 470 >33,000 VLX9hum_04 IgG4PE 256>33,000

TABLE 8 pH Dependent and Independent Binding of VLX4, VLX8 and VLX9Humanized mAbs. K_(D) (pM) K_(D) (pM) pH 6 pH 8 VLX9hum_03 IgG248 >33,000 VLX9hum_04 IgG2 43 >33,000 VLX9hum_06 IgG2 61 >33,000VLX9hum_08 IgG2 65 >33,000 VLX9hum_09 IgG2 138 >33,000 VLX4hum_07 IgG4PE63 92 VLX4hum_01 IgG4PE 47 75 VLX8hum_10 IgG4PE 52 79 VLX8hum_11 IgG4PE64 92

TABLE 9 pH Independent and Dependent Binding of VLX8hum_11 Fab andVLX9hum_08 Fab to Recombinant Human CD47 k_(a) k_(d) K_(D) (nM)VLX8hum_11 Fab 1.35e⁶ 2.29e⁻³ 1.7 nM (pH 7.5) VLX8hum_11 Fab 2.14e⁶2.78e⁻³ 1.3 nM (pH 6.5) VLX8hum_11 Fab 1.64e⁶ 2.63e⁻³ 1.6 nM (pH 6.0)VLX9hum_03 Fab 1.43e⁵ 1.13e⁻²  79 nM (pH 7.5) VLX9hum_08 Fab 1.74e⁵9.74e⁻⁴ 5.6 nM (pH 6.5) VLX9hum_08 Fab 1.95e⁵ 9.94e⁻⁴ 5.1 nM (pH 6.0)

TABLE 10 pH Dependent and Independent Binding of VLX4 and VLX9 HumanizedmAbs to Jurkat Cells KD (pM) KD (pM) pH 6.5 pH 7.4 VLX4hum_07 IgG4PE 6923 VLX9hum_04 IgG2 231 1526

Example 7 CD47 Antibodies Block CD47/SIRPα Binding

To assess the effect of humanized CD47 mAbs on binding of CD47 to SIRPαin vitro the following method is employed using the binding offluorescently-labelled SIRPα-Fc fusion protein to CD47 expressing Jurkatcells.

SIRPα-Fc fusion protein (R&D Systems, cat #4546-SA) was labelled usingan Alexa Fluor® antibody labelling kit (Invitrogen Cat No. A20186)according to the manufacturers specifications. 1.5×10⁶ Jurkat cells wereincubated with humanized mAbs (5 μg/ml), a human control antibody inRPMI containing 10% media or media alone for 30 min at 37° C. An equalvolume of fluorescently labelled SIRPα-Fc fusion protein was added andincubated for an additional 30 min at 37° C. Cells were washed once withPBS and the amount of labelled SIRPα-Fc bound to the Jurkat cellsanalyzed by flow cytometry.

As shown in FIG. 10, the humanized VLX4, VLX8 and VLX9 mAbs (VLX4hum_01IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_10 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_03 IgG2, VLX9hum_06 IgG2 and VLX9hum_08 IgG2) blocked theinteraction of CD47 expressed on the Jurkat cells with soluble SIPRα,while the human control antibody (which does not bind to CD47) or mediaalone, did not block the CD47/SIRPα interaction.

Example 8 CD47 Antibodies Increase Phagocytosis

To assess the effect of chimeric (murine-human) and humanized VLX4,VLX8, and VLX9 CD47 mAbs on phagocytosis of tumor cells by macrophagesin vitro the following method is employed using flow cytometry(Willingham et al. (2012) Proc Natl Acad Sci USA 109(17):6662-7 andTseng et al. (2013) Proc Natl Acad Sci USA 110(27): 11103-8).

Human derived macrophages were derived from leukapheresis of healthyhuman peripheral blood and incubated in AIM-V media (Life Technologies)for 7-10 days. For the in vitro phagocytosis assay, macrophages werere-plated at a concentration of 1×10⁴ cells per well in 100 ul of AIM-Vmedia in a 96-well plate and allowed to adhere for 24 hrs. Once theeffector macrophages adhered to the culture dish, the target humancancer cells (Jurkat) were labelled with 1 μM 5(6)-Carboxyfluoresceindiacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to themacrophage cultures at a concentration of 5×10⁴ cells in 1 ml of AIM-Vmedia (5:1 target to effector ratio). VLX4, VLX8, and VLX9 CD47 mAbs (1μg/ml) were added immediately upon mixture of target and effector cellsand allowed to incubate at 37° C. for 2-3 hours. After 2-3 hrs, allnon-phagocytosed cells were removed and the remaining cells washed threetimes with phosphate buffered saline (PBS; Sigma Aldrich). Cells werethen trypsinized, collected into microcentrifuge tubes, and incubated in100 ng of allophycocyanin (APC) labelled CD14 antibodies (BDBiosciences) for 30 minutes, washed once, and analyzed by flow cytometry(Accuri C6; BD Biosciences) for the percentage of CD14⁺ cells that werealso CFSE⁺ indicating complete phagocytosis.

As shown in FIG. 11, the VLX4 chimeric mAbs VLX4 IgG1 xi, VLX4 IgG1N297Q xi, VLX4 IgG4PE xi, and VLX4 IgG4 S228P xi increased phagocytosisof Jurkat cells by human macrophages by blocking the CD47/SIRPαinteraction. This enhanced phagocytosis is independent of Fc function.

Similarly, as shown in FIG. 12A and FIG. 12B, humanized mAbs VLX4hum_01IgG1, VLX4hum_01 IgG4PE, VLX4hum_06 IgG4PE, VLX4hum_07 IgG4PE,VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE increased phagocytosis ofJurkat cells by human macrophages by blocking the CD47/SIRPαinteraction. This enhanced phagocytosis is independent of Fc function.

As shown in FIG. 13A, the VLX8 chimeric mAbs VLX8 IgG1 N297Q xi and VLX8IgG4PE xi increase phagocytosis of Jurkat cells by human macrophages byblocking the CD47/SIRPα interaction. This enhanced phagocytosis isindependent of Fc function.

Similarly, as shown in FIG. 13B, humanized mAbs VLX8hum_01 IgG4PE,VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, VLX8hum_08 IgG4PE, and VLX8hum_09IgG4PE and chimeric mAb VLX8 IgG4PE xi increased phagocytosis of Jurkatcells by human macrophage by blocking the CD47/SIRPα interaction.

As shown in FIG. 14A, the VLX9 IgG1 N297Q xi, VLX9 IgG2 xi and VLX9IgG4PE xi chimeric mAbs all increased phagocytosis of Jurkat cells byhuman macrophages by blocking the CD47/SIRPα interaction. This enhancedphagocytosis is independent of Fc effector function. Similarly as shownin FIG. 14B, all of the humanized VLX9 IgG2 mAbs (VLX9hum_01 to _10IgG2) increased phagocytosis of Jurkat cells.

Example 9 Induction of Cell Death by Soluble CD47 Antibodies

Some soluble CD47 antibodies have been shown to induce selective celldeath of tumor cells. This additional property of selective toxicity tocancer cells is expected to have advantages compared to mAbs that onlyblock SIRPα binding to CD47.

Induction of cell death by soluble anti-CD47 mAbs is measured in vitro(Manna et al. (2003) J. Immunol. 107 (7): 3544-53). For the in vitrocell death assay, 1×10⁵ transformed human T cells (Jurkat cells) wereincubated with soluble humanized VLX4, VLX8, and VLX9 CD47 mAbs (1μg/ml) for 24 hrs at 37° C. As cell death occurs, mitochondrial membranepotential is decreased, the inner leaflet of the cell membrane isinverted, exposing phosphatidylserines (PS), and propidium iodide (PI)or 7-aminoactinomycin D (7-AAD) is able to incorporate into nuclear DNA.In order to detect these cellular changes, cells were then stained withfluorescently labelled annexin V and PI or 7-aminoactinomycin D (7-AAD)(BD Biosciences) and the signal detected using an Accuri C6 flowcytometer (BD Biosciences). The increase in PS exposure is determined bymeasuring the percent increase in annexin V signal and the percent ofdead cells by measuring the percent increase in PI or 7-AAD signal.Annexin V positive (annexin V⁺) or annexin V positive/7-AAD negative(annexin V⁺/7-AAD⁻) cells are observed in early stages of cell death andannexin V positive/7-AAD positive (annexin V⁺/7-AAD⁺) cells are deadcells. Importantly for therapeutic purposes, these mAbs induce celldeath of tumor cells directly and do not require complement or theintervention of other cells (e.g., NK cells, T cells, or macrophages) tokill. Thus, the mechanism is independent of both other cells and of Fceffector function. Therefore, therapeutic antibodies developed fromthese mAbs can be engineered to reduce Fc effector functions such asADCC and CDC and thereby limit the potential for side effects common tohumanized mAbs with intact Fc effector functions.

As shown in FIG. 15A-F, the soluble VLX4 humanized mAbs inducedincreased PS exposure and cell death of Jurkat cells as measured byincreased % of the cells that are annexin V⁺ (FIG. 15A and FIG. 15D),annexin V⁺/7-AAD⁻ (FIG. 15B and FIG. 15E), or annexin V⁺/7-AAD⁺ (FIG.15C and FIG. 15F). The humanized mAbs VLX4hum_01 IgG1, VLX4hum_01IgG4PE, VLX4hum_02 IgG1, VLX4hum_02 IgG4PE, VLX4hum_06 IgG4 PE,VLX4hum_07 IgG4PE, VLX4hum_12 IgG4PE, and VLX4hum_13 IgG4PE causedincreased PS exposure and cell death. In contrast, the humanized mAbsVLX4hum_08 IgG4PE and VLX4hum_11 IgG4PE did not cause increased PSexposure and cell death of Jurkat cells. Induction of cell death and thepromotion of phagocytosis of susceptible cancer cells imparts anadditional desirable antibody property and potential therapeutic benefitin the treatment of cancer.

As shown in FIGS. 16A-F, the soluble VLX8 chimeric and humanized mAbsinduced increased PS exposure and cell death of Jurkat cells as measuredby the % of the cells that are annexin V⁺ (FIGS. 16A, D), annexinV⁺/7-AAD⁻ (FIGS. 16B, E), or annexin V⁺/7-AAD⁺ (FIGS. 16C, F). Thechimeric mAbs, VLX8 IgG1 N297Q xi and VLX8 IgG4PE xi, and the humanizedmAbs, VLX8hum_07 IgG4PE and VLX8hum_08 IgG4PE, induced increased PSexposure and cell death of Jurkat cells. In contrast, the humanized mAbsVLX8hum_02 IgG4PE and VLX8hum_04 IgG4PE did not cause increased PSexposure and cell death of Jurkat cells. Induction of cell death and thepromotion of phagocytosis of susceptible cancer cells imparts anadditional desirable antibody property and potential therapeutic benefitin the treatment of cancer.

As shown in FIG. 17A-FIG. 17F, the soluble VLX9 chimeric and humanizedantibodies induced increased PS exposure and cell death of Jurkat cellsas measured by % of the cells that are annexin V⁺ (FIG. 17A and FIG.17D), annexin V⁺/7-AAD⁻ (FIG. 17B and FIG. 17E), or annexin V⁺/7-AAD⁺(FIG. 17C and FIG. 17F). The chimeric VLX9 IgG2xi mAb and the humanizedmAbs VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, and VLX9hum_09IgG2 induced increased PS exposure and cell death of Jurkat cells. Incontrast, the humanized mAbs VLX9hum_01 IgG2, VLX9hum_02 IgG2,VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2 and VLX9hum_010 IgG2did not cause increased PS exposure and cell death of Jurkat cells.Induction of cell death and the promotion of phagocytosis of susceptiblecancer cells imparts an additional desirable antibody property andpotential therapeutic benefit in the treatment of cancer. Importantly,chimeric and humanized mAbs that cause cell death of tumor cells do notcause cell death of normal cells.

Example 10 Damage-Associated Molecular Pattern (DAMP) Expression andRelease, Mitochondrial Depolarization and Cell Death Caused by HumanizedAnti-CD47 mAb

Humanized Anti-CD47 mAbs Cause Loss of Mitochondrial Membrane Potential

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure exhibit the ability to induce the loss ofmitochondrial membrane potential in tumor cell as described previously(Manna and Frazier, 2014 Journal of Immunology 170(7):3544-3553).

Loss of mitochondrial membrane potential in the tumor cell wasdetermined using JC-1 dye (Thermo; Catalogue #M34152). Human Rajilymphoma cells (ATCC, Manassas, Va.; Catalog # CCL-86) or other cellstypes that express sufficient levels of CD47 will be used. Cells weregrown in RPMI-1640 medium containing 10% (v/v) heat inactivated fetalbovine serum (BioWest; Catalogue # S01520), 100 units/mL penicillin, 100lag mL streptomycin (Sigma; Catalogue # P4222) at densities less than1×10⁶ cells/mL. For this assay, Raji cells were plated in 96 well tissueculture plates at a density of 1×10⁵ cells/ml RPMI-1640 mediumcontaining 10% (v/v) heat inactivated fetal bovine serum (BioWest;Catalog # S01520), 100 units/mL penicillin, 100 μg/mL streptomycin(Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2 VLX9hum_08 IgG2 and VLX9hum_03 IgG2)as disclosed herein, purified from transient transfections in CHO cellsas described above, as well as the control chimeric antibody, were addedat a final concentration of 10 μg/ml. As a positive control for loss ofmitochondrial membrane potential, cells were treated with 1 μM ofchemotherapeutic anthracycline mitoxantrone. The cells were incubated at37° C. for 24 hours, after which the cells were harvested, washed twicewith PBS, and incubated for 30 minutes with JC-1 dye as described above,diluted 1:2000 in PBS. After 30 minutes the cells were washed twice withPBS, resuspended in 100 μl of PBS, and analyzed for the percent of cellsthat shift their fluorescence emission from red to green by flowcytometry (Accuri C6, Becton Dickinson, Franklin Lakes, N.J.). Resultsare presented as means±SEM and analyzed for statistical significanceusing ANOVA in GraphPad Prism 6.

Some of the chimeric or humanized antibodies induce the loss ofmitochondrial membrane potential in the tumor cell. As shown in FIG. 18,the percent of cells with mitochondrial membrane depolarization in allanti-CD47 mAb treated cultures was significantly increased (p<0.05)compared to an isotype control. This increase in the amount ofmitochondrial membrane depolarization demonstrates that anti-CD47chimeric or humanized antibodies induce mitochondrial depolarizationthat leads to cell death in human tumor cells.

Humanized Anti-CD47 mAbs Cause Increase in Cell Surface CalreticulinExpression

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure exhibit the ability to expose the endoplasmicreticulum resident chaperone calreticulin on the surface of the tumorcell as, for example, described previously using chemotherapeuticanthracyclines such as doxorubicin and mitoxantrone, as disclosed byObeid et al. (2007) Nat. Med. 13(1):54-61.

Cell surface exposure of calreticulin was determined using a rabbitmonoclonal antibody against calreticulin conjugated to Alexa Fluor 647(Abcam; Catalogue #ab196159). Human Raji lymphoma cells (ATCC, Manassas,Va.; Catalog # CCL-86) or other cells types that express sufficientlevels of CD47 will be used. Cells were grown in RPMI-1640 mediumcontaining 10% (v/v) heat inactivated fetal bovine serum (BioWest;Catalogue # S01520), 100 units/mL penicillin, 100 lag mL streptomycin(Sigma; Catalogue # P4222) at densities less than 1×10⁶ cells/mL. Forthis assay, cells were plated in 96 well tissue culture plates at adensity of 1×10⁵ cells/ml RPMI-1640 medium containing 10% (v/v) heatinactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mLpenicillin, 100 μg/mL streptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 VLX9hum_03 IgG2) asdisclosed herein, purified from transient transfections in CHO cells asdescribed above, as well as the control chimeric antibody, were added ata final concentration of 10 μg/ml. As a positive control forcalreticulin exposure, cells were treated with 1 μM of chemotherapeuticanthracycline mitoxantrone. The cells were incubated at 37° C. for 24hours, after which the cells were harvested, washed twice with PBS, andincubated for 30 minutes with anti-calreticulin antibody as describedabove, diluted 1:200 in PBS. After 30 minutes the cells were washedtwice with PBS, resuspended in 100 μl of PBS, and analyzed for the meanfluorescence intensity of the anti-calreticulin antibody signal as wellas the percent of cells that stain positive for cell surfacecalreticulin by flow cytometry (Accuri C6, Becton Dickinson, FranklinLakes, N.J.). Results are presented as means±SEM and analyzed forstatistical significance using ANOVA in GraphPad Prism 6.

As shown in FIG. 19, the humanized antibodies induced the preapoptoticexposure of calreticulin on the tumor cell surface. The percent ofcalreticulin positive cells in all anti-CD47 mAb treated cultures wassignificantly increased (p<0.05) compared to an isotype control. Thisincrease in the exposure of calreticulin on the cell surfacedemonstrates that some of the humanized antibodies induce DAMPs fromtumor cells that can lead to phagocytosis of tumor cells and processingof tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increased Protein Disulfide-Isomerase 3(PDIA3) Expression

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure exhibit the ability to expose the endoplasmicreticulum resident chaperone PDIA3 on the surface of the tumor cell as,for example, described previously using chemotherapeutic anthracyclinessuch as doxorubicin and mitoxantrone, as disclosed by Panaretakis et al.(2008) Cell Death & Differentiation 15:1499-1509.

Cell surface exposure of PDIA3 was determined using a mouse monoclonalantibody against PDIA3 conjugated to FITC (Abcam; Catalogue #ab183396).Human Raji lymphoma cells (ATCC, Manassas, Va.; Catalog # CCL-86) orother cells types that express sufficient levels of CD47 will be used.Cells were grown in RPMI-1640 medium containing 10% (v/v) heatinactivated fetal bovine serum (BioWest; Catalogue # S01520), 100units/mL penicillin, 100 μg mL streptomycin (Sigma; Catalogue # P4222)at densities less than 1×10⁶ cells/mL. For this assay, cells were platedin 96 well tissue culture plates at a density of 1×10⁵ cells/mlRPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovineserum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 μg/mLstreptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)as disclosed herein, purified from transient transfections in CHO cellsas described above, as well as the control chimeric antibody, were addedat a final concentration of 10 μg/ml. As a positive control for PDIA3exposure, cells were treated with 1 μM of chemotherapeutic anthracyclinemitoxantrone. The Raji cells were incubated at 37° C. for 24 hours,after which the cells were harvested, washed twice with PBS, andincubated for 30 minutes with anti-PDIA3 antibody as described above,diluted 1:200 in PBS. After 30 minutes the cells were washed twice withPBS, resuspended in 100 μl of PBS, and analyzed for the meanfluorescence intensity of the anti-PDIA3 antibody signal as well as thepercent of cells that stain positive for cell surface calreticulin byflow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, N.J.).Results are presented as means±SEM and analyzed for statisticalsignificance using ANOVA in GraphPad Prism 6.

Some of the chimeric or humanized antibodies induce the preapoptoticexposure of PDIA3 on the tumor cell surface. As shown in FIG. 20, thepercent of PDIA3 positive cells in all the soluble anti-CD47 mAb treatedcultures was significantly increased (p<0.05) compared to the backgroundobtained with a negative control, humanized isotype-matched antibody.This increase in the exposure of PDIA3 on the cell surface demonstratesthat some of the chimeric or humanized antibodies induce DAMPs fromtumor cells that can lead to phagocytosis of tumor cells and processingof tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increased Cell Surface HSP70 Expression

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure exhibit the ability to expose the endoplasmicreticulum resident chaperone HSP70 on the surface of the tumor cell as,for example, described previously using chemotherapeutic anthracyclinessuch as doxorubicin and mitoxantrone, as disclosed by Fucikova et al.(2011) Cancer Research 71(14):4821-4833.

Cell surface exposure of HSP70 was determined using a mouse monoclonalantibody against HSP70 conjugated to Phycoerythrin (Abcam; Catalogue#ab65174). Human Raji lymphoma cells (ATCC, Manassas, Va.; Catalog #CCL-86) or other cells types that express sufficient levels of CD47 wereused. Cells were grown in RPMI-1640 medium containing 10% (v/v) heatinactivated fetal bovine serum (BioWest; Catalogue # S01520), 100units/mL penicillin, 100 μg mL streptomycin (Sigma; Catalogue # P4222)at densities less than 1×10⁶ cells/mL. For this assay, cells were platedin 96 well tissue culture plates at a density of 1×10⁵ cells/mlRPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovineserum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 μg/mLstreptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)as disclosed herein, purified from transient transfections in CHO cellsas described above, as well as the control chimeric antibody, were addedat a final concentration of 10 μg/ml. As a positive control for HSP70exposure, Raji cells were treated with 1 μM of chemotherapeuticanthracycline mitoxantrone. The cells were incubated at 37° C. for 24hours, after which the cells were harvested, washed twice with PBS, andincubated for 30 minutes with anti-HSP70 antibody as described above,diluted 1:200 in PBS. After 30 minutes the cells were washed twice withPBS, resuspended in 100 μl of PBS, and analyzed for the meanfluorescence intensity of the anti-HSP70 antibody signal as well as thepercent of cells that stain positive for cell surface calreticulin byflow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, N.J.).Results are presented as means±SEM and analyzed for statisticalsignificance using ANOVA in GraphPad Prism 6.

Some of the chimeric or humanized antibodies induce the preapoptoticexposure of HSP70 on the tumor cell surface. As shown in FIG. 21, thepercent of HSP70 positive cells in all anti-CD47 mAb treated cultureswas significantly increased (p<0.05) compared to those seen in isotypecontrol treated cultures. This increase in the exposure of HSP70 on thecell surface demonstrates that some of the chimeric or humanizedantibodies induce DAMPs from tumor cells and can lead to phagocytosis oftumor cells and processing of tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increased Cell Surface HSP90 Expression

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure expose the endoplasmic reticulum resident chaperoneHSP70 on the surface of the tumor cell as, for example, describedpreviously using chemotherapeutic anthracyclines such as doxorubicin andmitoxantrone, as disclosed by Fucikova et al. (2011) Cancer Research71(14):4821-4833.

Cell surface exposure of HSP90 was determined using a mouse monoclonalantibody against HSP70 conjugated to Phycoerythrin (Abcam; Catalogue#ab65174). Human Raji lymphoma cells (ATCC, Manassas, Va.; Catalog #CCL-86) or other cells types that express sufficient levels of CD47 wereused. Cells are grown in RPMI-1640 medium containing 10% (v/v) heatinactivated fetal bovine serum (BioWest; Catalogue # S01520), 100units/mL penicillin, 100 μg mL streptomycin (Sigma; Catalogue # P4222)at densities less than 1×10⁶ cells/mL. For this assay, cells were platedin 96 well tissue culture plates at a density of 1×10⁵ cells/mlRPMI-1640 medium containing 10% (v/v) heat inactivated fetal bovineserum (BioWest; Catalog # S01520), 100 units/mL penicillin, 100 μg/mLstreptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)as disclosed herein, purified from transient transfections in CHO cellsas described above, as well as the control chimeric antibody, were addedat a final concentration of 10 μg/ml. As a positive control for HSP90exposure, cells were treated with 1 μM of chemotherapeutic anthracyclinemitoxantrone. The Raji cells were incubated at 37° C. for 24 hours,after which the cells were harvested, washed twice with PBS, andincubated for 30 minutes with anti-HSP70 antibody as described above,diluted 1:200 in PBS. After 30 minutes the cells were washed twice withPBS, resuspended in 100 μl of PBS, and analyzed for the meanfluorescence intensity of the anti-HSP70 antibody signal as well as thepercent of cells that stain positive for cell surface calreticulin byflow cytometry (Accuri C6, Becton Dickinson, Franklin Lakes, N.J.).Results are presented as means±SEM and analyzed for statisticalsignificance using ANOVA in GraphPad Prism 6.

Some of the chimeric or humanized antibodies induce the preapoptoticexposure of HSP90 on the tumor cell surface. As shown in FIG. 22, thepercent of HSP90 positive cells in soluble anti-CD47 mAb-treatedcultures was significantly increased (p<0.05) compared to the backgroundobtained with a negative control, humanized isotype-matched antibody,except for VLXhum_06 IgG2 and VLX4hum_01 IgG4PE (ns, not significant).This increase in the exposure of HSP90 on the cell surface demonstratesthat some of the chimeric or humanized antibodies induce DAMPs fromtumor cells and can lead to phagocytosis of tumor cells and processingof tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increased ATP Release

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure induce increased release of adenosine triphosphate(ATP) from the tumor cell as described previously using anthracyclinechemotherapy drugs (Martins et al., 2014 Cell Death and Differentiation21:79-91).

Release of ATP from the tumor cell is determined by quantitativebioluminescence assay as described by the manufacturer (MolecularProbes; Catalogue #A22066). Human Raji lymphoma cells (ATCC, Manassas,Va.; Catalog # CCL-86) or other cells types that express sufficientlevels of CD47 were used. Cells were grown in RPMI-1640 mediumcontaining 10% (v/v) heat inactivated fetal bovine serum (BioWest;Catalogue # S01520), 100 units/mL penicillin, 100 μg mL streptomycin(Sigma; Catalogue # P4222) at densities less than 1×10⁶ cells/mL. Forthis assay, cells were plated in 96 well tissue culture plates at adensity of 1×10⁵ cells/ml RPMI-1640 medium containing 10% (v/v) heatinactivated fetal bovine serum (BioWest; Catalog # S01520), 100 units/mLpenicillin, 100 μg/mL streptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03) asdisclosed herein, purified from transient transfections in CHO cells asdescribed above, as well as the control chimeric antibody, were added ata final concentration of 10 μg/ml. As a positive control for ATPrelease, cells were treated with 1 μM of chemotherapeutic anthracyclinemitoxantrone. The cells were incubated at 37° C. for 24 hours, afterwhich the cell-free supernatant was collected and stored at −80° C.After all samples have been collected, 10 μl of each sample was testedby the ATP determination kit as described above. Final concentrationswere determined by comparing experimental values to a standard curve anddisplayed as the concentration of ATP (μM) released by tumor cells inresponse to antibody treatment. Results are presented as means±SEM andanalyzed for statistical significance using ANOVA in GraphPad Prism 6.

The humanized antibodies increased the release of ATP from the tumorcells. As shown in FIG. 23, the amount of released ATP in all anti-CD47mAb treated cultures was significantly increased (p<0.05) compared to anisotype control. This increase in the release of ATP demonstrates thatsome of the chimeric or humanized antibodies induce the release of ATPfrom tumor cells and can lead to dendritic cell migration through itscognate purinergic receptors.

Humanized Anti-CD47 mAbs Cause HMGB1 Release

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure increase the release of the non-histone chromatinprotein high-mobility group box 1 (HMGB1) from the tumor cell asdescribed previously using chemotherapy agents, such as oxaliplatin(Tesniere et al., 2010 Oncogene, 29:482-491) and mitoxantrone (Michaudet al., 2011 Science 334:1573-1577).

Release of HMGB1 protein from the tumor cell was determined by enzymeimmunoassay as described by the manufacturer (IBL International;Hamburg, Germany, Catalogue #ST51011). Human Raji lymphoma cells (ATCC,Manassas, Va.; Catalog # CCL-86) or other cells types that expresssufficient levels of CD47 were used. Cells will be grown in RPMI-1640medium containing 10% (v/v) heat inactivated fetal bovine serum(BioWest; Catalogue # S01520), 100 units/mL penicillin, 100 μg mLstreptomycin (Sigma; Catalogue #P4222) at densities less than 1×10⁶cells/mL. For this assay, cells were plated in 96 well tissue cultureplates at a density of 1×10⁵ cells/ml RPMI-1640 medium containing 10%(v/v) heat inactivated fetal bovine serum (BioWest; Catalog # S01520),100 units/mL penicillin, 100 μg/mL streptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)as disclosed herein, purified from transient transfections in CHO cellsas described above, as well as the control chimeric antibody, will thenbe added at a final concentration of 10 μg/ml.

As a positive control for HMGB1 release, Raji cells were treated with 1μM of chemotherapeutic anthracycline mitoxantrone. The cells wereincubated at 37° C. for 24 hours, after which the cell-free supernatantwas collected and stored at −80° C. After all samples have beencollected, 10 μl of each sample was tested by HMGB1 ELISA as describedabove. Final concentrations were determined by comparing experimentalvalues to a standard curve and reported as the concentration of HMGB1(ng/ml) released by tumor cells in response to antibody treatment.Results are presented as means±SEM and analyzed for statisticalsignificance using ANOVA in GraphPad Prism 6.

As shown in FIG. 24, the humanized antibodies increased the release ofHMGB1 protein from the tumor cells. The amount of released HMGB1 proteinin all anti-CD47 mAb treated cultures was significantly increased(p<0.05) compared to an isotype control, except for VLX9hum_06 IgG2 (ns,not significant). This increase in the release of HMGB1 demonstratesthat some of the chimeric or humanized antibodies induce release ofDAMPs from tumor cells and can lead to dendritic cell activation.

Humanized Anti-CD47 mAbs Cause CXCL10 Release

These experiments demonstrate that humanized anti-CD47 mAbs of thepresent disclosure increase the production and release of the chemokineCXCL10 from the human tumor cells as described previously usinganthracycline chemotherapy drugs (Sistigu et al., 2014 Nat. Med.20(11):1301-1309).

Release of the CXCL10 from the tumor cell was determined by enzymeimmunoassay as described by the manufacturer (R&D Systems; Catalogue#DIP100). Human Raji lymphoma cells (ATCC, Manassas, Va.; Catalog #CCL-86) or other cells types that express sufficient levels of CD47 willbe used. Cells were grown in RPMI-1640 medium containing 5% (v/v) heatinactivated fetal bovine serum (BioWest; Catalogue # S01520), 100units/mL penicillin, 100 μg mL streptomycin (Sigma; Catalogue # P4222)at densities less than 1×10⁶ cells/mL. For this assay, cells were platedin 96 well tissue culture plates at a density of 1×10⁵ cells/mlRPMI-1640 medium containing 5% (v/v) heat inactivated fetal bovine serum(BioWest; Catalog # S01520), 100 units/mL penicillin, 100 μg/mLstreptomycin (Sigma; #P4222).

The humanized antibodies (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE,VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2)as disclosed herein, purified from transient transfections in CHO cellsas described above, as well as the control chimeric antibody, were addedat a final concentration of 10 μg/ml. As a positive control for CXCL10release, Raji cells were treated with 1 μM of the chemotherapeuticanthracycline mitoxantrone. The cells were incubated at 37° C. for 24hours, after which the cell-free supernatant was collected and stored at−80° C. After all samples have been collected, 10 μl of each sample wastested by the CXCL10 ELISA as described above. Final concentrations weredetermined by comparing experimental values to a standard curve anddisplayed as the concentration of CXCL10 (pg/ml) released by tumor cellsin response to antibody treatment.

Some of the chimeric or humanized antibodies induce release of CXCL10 byhuman tumor cells. As shown in FIG. 25, the amount of released CXCL10 inall anti-CD47 mAb treated cultures significantly increased (p<0.05)compared to an isotype control. This increase in the release of CXCL10demonstrates that some of the chimeric or humanized antibodies inducethe release of CXCL10 from tumor cells and suggest a role in therecruitment of immune cells to the tumor.

Example 11 Damage-Associated Molecular Pattern (DAMP) Expression andRelease, Mitochondrial Depolarization and Cell Death Caused by HumanizedAnti-CD47 mAbs

These studies were conducted as described in Example 10, except that thehuman Jurkat T ALL cell line (ATCC, Manassas, Va.; Catalog # TIB-152)was used.

Humanized Anti-CD47 mAbs Cause Loss of Mitochondrial Membrane Potential

As shown in FIG. 26, the humanized mAbs (VLX4hum_01 IgG4PE, VLX4hum_07IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2 andVLX9hum_03 IgG2) caused a significant increase in the percent of cellswith mitochondrial membrane depolarization (p<0.05) compared to anisotype control. This increase in the amount of mitochondrial membranedepolarization demonstrates that some of the chimeric or humanizedantibodies induce cell death in human tumor cells.

Humanized Anti-CD47 mAbs Cause Increase in Cell Surface CalreticulinExpression

As shown in FIG. 27, the humanized antibodies (VLX4hum_01 IgG4PE,VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06 IgG2, VLX9hum_08 IgG2and VLX9hum_03 IgG2) induced the preapoptotic exposure of calreticulinon the tumor cell surface. The percent of calreticulin positive cells inall anti-CD47 mAb treated cultures were significantly increased (p<0.05)compared to an isotype control, except VLX9hum_03 IgG2 (ns). Thisincrease in the exposure of calreticulin on the cell surfacedemonstrated that some of the humanized antibodies induce DAMPs fromtumor cells and can lead to phagocytosis of tumor cells and processingof tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increase in Cell Surface PDIA3 Expression

As shown in FIG. 28, the percent of PDIA3 positive cells in solubleanti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultureswere significantly increased (p<0.05) compared to the backgroundobtained with a negative control, humanized isotype-matched antibody.This increase in the exposure of PDIA3 on the cell surface demonstratesthat some of the chimeric or humanized antibodies induce DAMPs fromtumor cells and can lead to phagocytosis of tumor cells and processingof tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increase in Cell Surface HSP70 Expression

As shown in FIG. 29, the percent of HSP70 positive cells in anti-CD47mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures weresignificantly increased (p<0.05) compared to those seen in isotypecontrol treated cultures. Although each of the anti-CD47 mAbs caused astatistically significant increase in HSP70 expression, mitoxantrone didnot. This increase in the exposure of HSP70 on the cell surfacedemonstrates that some of the chimeric or humanized antibodies induceDAMPs from tumor cells and can lead to phagocytosis of tumor cells andprocessing of tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increase in Cell Surface HSP90 Expression

As shown in FIG. 30, the percent of HSP90 positive cells in solubleanti-CD47 mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE,VLX9hum_06 IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultureswere significantly increased (p<0.05) compared to the backgroundobtained with a negative control, humanized isotype-matched antibody.This increase in the exposure of HSP90 on the cell surface demonstratesthat some of the chimeric or humanized antibodies induce DAMPs fromtumor cells and can lead to phagocytosis of tumor cells and processingof tumor antigen by innate immune cells.

Humanized Anti-CD47 mAbs Cause Increase in ATP Release

As shown in FIG. 31, the amount of released ATP in humanized anti-CD47mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures wassignificantly increased (p<0.05) compared to an isotype control.Although each of the anti-CD47 mAbs caused a statistically significantincrease in HSP70 expression, mitoxantrone did not (ns). This increasein the release of ATP will demonstrates that some of the chimeric orhumanized antibodies induce the release of ATP from tumor cells and canlead to dendritic cell migration through its cognate purinergicreceptors.

Humanized Anti-CD47 mAbs Cause Increase in HMGB1 Release

As shown in FIG. 32, the amount of released HMGB1 protein in anti-CD47mAb (VLX4hum_01 IgG4PE, VLX4hum_07 IgG4PE, VLX8hum_11 IgG4PE, VLX9hum_06IgG2, VLX9hum_08 IgG2 and VLX9hum_03 IgG2) treated cultures wassignificantly increased (p<0.05) compared to an isotype control, exceptfor VLX4hum_01 IgG4PE (ns). This increase in the release of HMGB1demonstrates that some of the chimeric or humanized antibodies induceDAMPs from tumor cells and can lead to dendritic cell activation.

Example 12 Hemagglutination of Human Red Blood Cells (hRBCs)

Many CD47 antibodies, including B6H12, BRIC126, MABL1, MABL2, CC2C6,5F9, have been shown to cause hemagglutination (HA) of washed RBCs invitro or in vivo (Petrova P. et al. Cancer Res 2015; 75(15 Suppl):Abstract nr 4271; U.S. Pat. No. 9,045,541; Uno et al. Oncol Rep. 17:1189-94, 2007; Kikuchi et al. Biochem Biophys Res. Commun. 315: 912-8,2004; Sikic B. et al. J Clin Oncol 2016; 34 (suppl; abstract 3019)).Hemagglutination of hRBCs was assessed following incubation of hRBCswith various concentrations of chimeric and humanized VLX4, VLX8, andVLX9 mAbs in vitro essentially as described by Kikuchi et al. BiochemBiophys Res. Commun (2004) 315:912-918. Blood was obtained from healthydonors, diluted (1:50) in PBS/1 mM EDTA/BSA and washed 3 times withPBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equalvolumes of the antibodies (75 μl of each) and incubated for 3 hrs at 37°C. and overnight at 4° C. A tight RBC pellet is observed with antibodiesthat do not cause hemagglutination, and a diffuse, hazy pattern isobserved with antibodies that cause hemagglutination.

As shown in FIG. 33A and Tables 1 and 2, The VLX4hum_01 IgG1 causedvisible hemagglutination of hRBCs, whereas the humanized VLX4hum_01IgG4PE mAb did not (mAb concentrations 50 μg/ml to 0.3 ng/ml). The lackof detectable hemagglutination by VLX4hum_01 IgG4 PE imparts anadditional desirable antibody property and potential therapeutic benefitin the treatment of cancer.

As shown in FIG. 33B and Tables 1 and 2, the chimeric antibody VLX8IgG4PE (xi) and the humanized antibodies VLX8hum_08 IgG4PE, VLX8hum_09IgG4PE, and VLX8hum_10 IgG4PE caused visible hemagglutination of hRBCs,whereas the VLX8 humanized Abs VLX8hum_01 IgG4PE, VLX8hum_02 IgG4 PE,VLX8hum_03 IgG4 PE and VLX8hum_11 IgG4PE did not (mAb concentrations 50μg/ml to 0.3 ng/ml).

The lack of detectable hemagglutination by humanized antibodiesVLX4hum_01 IgG4PE, VLX8hum_01 IgG4PE, VLX8hum_02 IgG4 PE, VLX8hum_03IgG4 PE and VLX8hum_11 IgG4 PE imparts an additional desirable antibodyproperty and a potential therapeutic benefit in the treatment of cancer.

As shown in FIG. 34A and FIG. 34B, the chimeric antibody VLX9 IgG2 xicaused visible hemagglutination of hRBCs, whereas all of the humanizedVLX9 mAbs except for VLX9hum_07 IgG2, did not cause detectablehemagglutination (at concentrations from 50 ug/ml to 0.3 μg/ml).However, the amount of detectable hemagglutination caused by VLX9hum_07was reduced compared to the VLX9 IgG2 chimeric mAb. Again, the reducedor lack of detectable hemagglutination by the VLX9 humanized mAbsimparts an additional desirable antibody property and a potentialtherapeutic benefit in the treatment of cancer.

Example 13 Anti-Tumor Activity In Vivo

The purpose of this experiment was to demonstrate that VLX4, VLX8 andVLX9 humanized antibodies, exemplified by VLX4_07 IgG4PE, VLX8_10 IgG4PEand VLX9hum_08 IgG2, reduce tumor burden in vivo in a mouse xenograftmodel of lymphoma.

Raji human Burkitt's lymphoma cells (ATCC #CCL-86, Manassas, Va.) weremaintained in RPMI-1640 (Lonza; Walkersville, Md.) supplemented with 10%Fetal Bovine Serum (FBS; Omega Scientific; Tarzana, Calif.) within a 5%CO₂ atmosphere. Cultures were expanded in tissue culture flasks.

Female NSG (NOD-Cg-Prkdc^(scid)I12rg^(tm1Wjl)/SzJ) were obtained fromJackson Laboratory (Bar Harbor, Me.) at 5-6 weeks of age. Mice wereacclimated prior to handling and housed in microisolator cages (LabProducts, Seaford, Del.) under specific pathogen-free conditions. Micewere fed Teklad Global Diet® 2920x irradiated laboratory animal diet(Envigo, Formerly Harlan; Indianapolis, Ind.) and provided autoclavedwater ad libitum. All procedures were carried out under InstitutionalAnimal Care and Use guidelines.

Female NSG mice were inoculated subcutaneously in the right flank with0.1 mL of a 30% RPMI/70% Matrigel™ (BD Biosciences; Bedford, Mass.)mixture containing a suspension of 5×10⁶ Raji tumor cells. Five daysfollowing inoculation, digital calipers were used to measure width andlength diameters of the tumor. Tumor volumes were calculated utilizingthe formula: tumor volume (mm³)=(a×b²/2) where ‘b’ is the smallestdiameter and ‘a’ is the largest diameter. Mice with palpable tumorvolumes of 31-74 mm³ were randomized into 8-10/group and VLX9hum_08 orPBS (control) administration was initiated at this time. Mice weretreated with 5 mg/kg of antibody 5×/week for 4 weeks by intraperitonealinjection. Tumor volumes and body weights were recorded twice weekly.

As shown in FIG. 35, treatment with the humanized VLX4hum_07 IgG4PEsignificantly reduced tumor growth of the Raji tumors (p<0.05, two-wayANOVA), demonstrating anti-tumor efficacy in vivo.

As shown in FIG. 36, treatment with the humanized anti-CD47 mAb,VLX8hum_10 IgG4PE significantly reduced (p<0.0001, two-way ANOVA) tumorgrowth of the Raji tumors, demonstrating anti-tumor efficacy in vivo.

As shown in FIG. 37, treatment with the humanized anti-CD47 mAb,VLX9hum_08 IgG2 significantly reduced (p<0.05, two-way ANOVA) tumorgrowth of the Raji tumors, demonstrating anti-tumor efficacy in vivo.

Example 14 Effect on Circulating Red Blood Cell Parameters

The purpose of this experiment is to demonstrate that VLX9 humanizedantibodies that do not bind to human RBC in vitro (Table 2), exemplifiedby hum1017_08 IgG2, do not cause a reduction in either hemoglobin (Hg)or circulating RBCs following administration to cynomolgus monkeys.

Female Chinese cynomolgus monkeys (Charles River Laboratories, Houston,Tex.) 2.5-3 kg were used in accordance with the Institutional AnimalCare and Use guidelines. VLX9hum_08 IgG2 or vehicle (PBS) wasadministered as a 1 hour intravenous infusion on day 1 at a dose of 5mg/kg and on day 18 at a dose of 15 mg/kg (3 animals/group).Hematological parameters were measured throughout the study on days −7,−3 (not shown), pre-dose, 3, 8, 12, 18 (pre-dose), 20, 25, 29, 35 and 41and compared/normalized to the means values of control animals. Thepre-treatment RBC and Hg values on day 0 in the VLX9hum_08 IgG2 groupwere lower than the control group. Following treatment with either doseof VLX9hum_08 IgG2, there were minimal changes (<10%) in Hg (FIG. 38A)or RBC counts (FIG. 38B) compared to the control group demonstratingthat VLX9hum_08 IgG2 causes minimal reductions in RBC hematologicalparameters when administered to cynomolgus monkeys.

Example 15 Antibodies to CD47 Regulate Nitric Oxide Signaling

TSP1 binding to CD47 activates the heterotrimeric G protein Gi, whichleads to suppression of intracellular cyclic AMP (cAMP) levels. Inaddition, the TSP1/CD47 pathway opposes the beneficial effects of thenitric oxide (NO) pathway in all vascular cells. The NO pathway consistsof any of three nitric oxide synthase enzymes (NOS I, NOS II and NOSIII) that generate bioactive gas NO using arginine as a substrate. NOcan act within the cell in which it is produced or in neighboring cells,to activate the enzyme soluble guanylyl cyclase that produces themessenger molecule cyclic GMP (cGMP). The proper functioning of theNO/cGMP pathway is essential for protecting the cardiovascular systemagainst stresses including, but not limited to, those resulting fromwounding, inflammation, hypertension, metabolic syndrome, ischemia, andischemia-reperfusion injury (IRI). In the context of these cellularstresses, the inhibition of the NO/cGMP pathway by the TSP1/CD47 systemexacerbates the effects of stress. This is a particular problem in thecardiovascular system where both cGMP and cAMP play important protectiveroles. There are many cases in which ischemia and reperfusion injurycause or contribute to disease, trauma, and poor outcomes of surgicalprocedures.

The purpose of these experiment will be to demonstrate that humanizedanti-CD47 mAbs of the present disclosure exhibit the ability to reverseTSP1-mediated inhibition of NO-stimulated cGMP synthesis as, forexample, described previously using mouse monoclonal antibodies to CD47as disclosed by Isenberg et al. (2006) J. Biol. Chem. 281:26069-80, oralternatively other downstream markers of or effects resulting from NOsignaling, for example smooth muscle cell relaxation or plateletaggregation as described previously by Miller et al. (2010) Br J.Pharmacol. 159: 1542-1547.

The method employed that will be to measure cGMP as described by themanufacturer (CatchPoint Cyclic-GMP Fluorescent Assay Kit, MolecularDevices, Sunnyvale, Calif.). Jurkat JE6.1 cells (ATCC, Manassas, Va.;Catalog # TIB-152) or other cells types that retain the NO/cGMPsignaling pathway when grown in culture and exhibit a robust andreproducible inhibitory response to TSP1 ligation of CD47 will be used.Cells will be grown in Iscove's modified Dulbeccco's medium containing5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue #S01520), 100 units/mL penicillin, 100 ag mL streptomycin (Sigma;Catalogue # P4222) at densities less than 1×106 cells/mL. For the cGMPassay, cells will be plated in 96 well tissue culture plates at adensity of 1×10⁵ cells/ml in Iscoves modified Dulbecco's mediumcontaining 5% (v/v) heat inactivated fetal bovine serum (BioWest;Catalog # S01520), 100 units/mL penicillin, 100 μg/mL streptomycin(Sigma; #P4222) for 24 hours and then transferred to serum free mediumovernight.

The humanized antibodies as disclosed herein, purified from transienttransfections in CHO cells as described above in Example 3, as well asthe control chimeric antibody, will then be added at a finalconcentration of 20 ng/ml, followed 15 minutes later by 0 or 1 μg/mlhuman TSP1 (Athens Research and Technology, Athens, Ga., Catalogue#16-20-201319). After an additional 15 minutes, the NO donor,diethylamine (DEA) NONOate (Cayman Chemical, Ann Arbor, Mich., Catalog#82100), will be added to half the wells at a final concentration of 1μM. Five minutes later, the cells will be lysed with buffer supplied inthe cGMP kit, and aliquots of each well assayed for cGMP content.

It is anticipated that some of the chimeric or humanized antibodies willreverse TSP1 inhibition of cGMP. Reversal will be complete (>80%) orintermediate (20%-80%). This reversal of TSP1 inhibition of cGMP willdemonstrate that they have the ability to increase NO signaling andsuggest utility in protecting the cardiovascular system against stressesincluding, but not limited to, those resulting from wounding,inflammation, hypertension, metabolic syndrome, ischemia, andischemia-reperfusion injury (IRI). Additional assay systems, for examplesmooth muscle cell contraction, will also be expected to show that someof the chimeric or humanized antibody clones reverse the inhibitoryactions of TSP on downstream effects resulting from the activation of NOsignaling.

1-101. (canceled)
 102. A monoclonal antibody, or an antigen bindingfragment thereof, which: a. binds to human CD47; b. blocks SIRP (bindingto human CD47; c. increases phagocytosis of human tumor cells; d.induces death of human tumor cells; e. exhibits pH-dependent binding tohuman CD47 present on a cell; and wherein the monoclonal antibody, or anantigen binding fragment thereof, further comprises one or morecharacteristics selected from the group consisting of: i) causes nodetectable agglutination of human red blood cells, or causes reducedagglutination of human red blood cells, and ii) has reduced human redblood cell binding, or has minimal human red blood cell binding. 103.The monoclonal antibody, or antigen binding fragment thereof, of claim102, wherein said monoclonal antibody, or an antigen binding fragmentthereof, possesses one or more among the following characteristics:causes an increase in cell surface calreticulin expression on humantumor cells; causes an increase in adenosine triphosphate (ATP) releaseby human tumor cells; causes an increase in high mobility group box 1(HMGB1) release by human tumor cells; causes an increase in annexin A1release by human tumor cells; causes an increase in Type I Interferonrelease by human tumor cells; causes an increase in C-X-C MotifChemokine Ligand 10 (CXCL10) release by human tumor cells; causes anincrease in cell surface protein disulfide-isomerase A3 (PDIA3)expression on human tumor cells; causes an increase in cell surface heatshock protein 70 (HSP70) expression on human tumor cells; and causes anincrease in cell surface heat shock protein 90 (HSP90) expression onhuman tumor cells.
 104. The monoclonal antibody, or antigen bindingfragment thereof, of claim 102, wherein the antibody, or antigen bindingfragment thereof, is a chimeric or humanized antibody; or wherein themonoclonal antibody, or antigen binding fragment thereof cross-reactswith one or more species homologs of CD47.
 105. The monoclonal antibody,or antigen binding fragment thereof, of claim 102, wherein the antibody,or antigen binding fragment thereof, causes no detectable agglutinationof human red blood cells and has minimal human red blood cell binding.106. The monoclonal antibody, or antigen binding fragment thereof, ofclaim 102, wherein the pH-dependent binding to human CD47 has a greateraffinity for human CD47 at an acidic pH compared to physiological pH.107. A method of treating a disease comprising an autoimmune disease, anautoinflammatory disease, an inflammatory disease, a cardiovasculardisease, or a cancer in a subject, comprising administration of themonoclonal antibody or antigen-binding fragment thereof, of claim 102,wherein the subject comprises a human or companion/pet animal, a workinganimal, a sport animal, a zoo animal, or a valuable animal kept incaptivity.
 108. The method of claim 107, wherein the disease is selectedfrom the group consisting of a leukemia, a lymphoma, ovarian cancer,breast cancer, endometrial cancer, colon cancer (colorectal cancer),rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-smallcell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma ofthe lung), bronchial cancer, bone cancer, prostate cancer, pancreaticcancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer,bile duct cancer, esophageal cancer, renal cell carcinoma, thyroidcancer, squamous cell carcinoma of the head and neck (head and neckcancer), testicular cancer, cancer of the endocrine gland, cancer of theadrenal gland, cancer of the pituitary gland, cancer of the skin, cancerof soft tissues, cancer of blood vessels, cancer of brain, cancer ofnerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancerof hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma,meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma,neuroblastoma, melanoma, myelodysplastic syndrome, a sarcoma,ischemia-reperfusion injury, heart failure, arthritis, rheumatoidarthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, lupus, systemiclupus erythematous, juvenile rheumatoid arthritis, juvenile idiopathicarthritis, Grave's disease, Hashimoto's thyroiditis, Addison's disease,celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis,pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,uveitis, atherosclerosis, and ankylosing spondylitis.
 109. A monoclonalantibody, or an antigen binding fragment thereof, which: a. binds tohuman CD47; b. blocks SIRPα binding to human CD47; c. increasesphagocytosis of human tumor cells; d. induces death of human tumorcells; e. exhibits pH-dependent binding to human CD47 present on a cell;f. exhibits reduced binding to a normal cell; and wherein the monoclonalantibody, or an antigen binding fragment thereof, further comprises oneor more characteristics selected from the group consisting of: i) causesno detectable agglutination of human red blood cells, or causes reducedagglutination of human red blood cells; and ii) has reduced human redblood cell binding, or has minimal human red blood cell binding. 110.The monoclonal antibody, or antigen binding fragment thereof, of claim109, wherein said monoclonal antibody, or an antigen binding fragmentthereof, further comprises one or more characteristics selected from thegroup consisting of: causes an increase in cell surface calreticulinexpression on human tumor cells; causes an increase in adenosinetriphosphate (ATP) release by human tumor cells; causes an increase inhigh mobility group box 1 (HMGB1) release by human tumor cells; causesan increase in annexin A1 release by human tumor cells; causes anincrease in Type I Interferon release by human tumor cells; causes anincrease in C-X-C Motif Chemokine Ligand 10 (CXCL10) release by humantumor cells; causes an increase in cell surface proteindisulfide-isomerase A3 (PDIA3) expression on human tumor cells; causesan increase in cell surface heat shock protein 70 (HSP70) expression onhuman tumor cells; and causes an increase in cell surface heat shockprotein 90 (HSP90) expression on human tumor cells.
 111. The monoclonalantibody, or antigen binding fragment thereof, of claim 109, wherein theantibody, or antigen binding fragment thereof, is a chimeric orhumanized antibody; or wherein the monoclonal antibody, or antigenbinding fragment thereof cross-reacts with one or more species homologsof CD47.
 112. The monoclonal antibody, or antigen binding fragmentthereof, of claim 109, wherein the antibody, or antigen binding fragmentthereof, causes no detectable agglutination of human red blood cells andhas minimal human red blood cell binding.
 113. The monoclonal antibody,or antigen binding fragment thereof, of claim 109, wherein thepH-dependent binding to CD47 has a greater affinity for CD47 at anacidic pH compared to physiological pH.
 114. A method of treating adisease comprising an autoimmune disease, an autoinflammatory disease,an inflammatory disease, a cardiovascular disease, or a cancer in asubject, comprising administration of the monoclonal antibody orantigen-binding fragment thereof, of claim 109, wherein subjectcomprises a human or companion/pet animal, a working animal, a sportanimal, a zoo animal, or a valuable animal kept in captivity.
 115. Themethod of claim 114, wherein the disease is selected from the groupconsisting of a leukemia, a lymphoma, ovarian cancer, breast cancer,endometrial cancer, colon cancer (colorectal cancer), rectal cancer,bladder cancer, urothelial cancer, lung cancer (non-small cell lungcancer, adenocarcinoma of the lung, squamous cell carcinoma of thelung), bronchial cancer, bone cancer, prostate cancer, pancreaticcancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer,bile duct cancer, esophageal cancer, renal cell carcinoma, thyroidcancer, squamous cell carcinoma of the head and neck (head and neckcancer), testicular cancer, cancer of the endocrine gland, cancer of theadrenal gland, cancer of the pituitary gland, cancer of the skin, cancerof soft tissues, cancer of blood vessels, cancer of brain, cancer ofnerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancerof hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma,meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma,neuroblastoma, melanoma, myelodysplastic syndrome, a sarcoma,ischemia-reperfusion injury, heart failure, arthritis, rheumatoidarthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, lupus, systemiclupus erythematous, juvenile rheumatoid arthritis, juvenile idiopathicarthritis, Grave's disease, Hashimoto's thyroiditis, Addison's disease,celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis,pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,uveitis, atherosclerosis, and ankylosing spondylitis.
 116. A monoclonalantibody, or an antigen binding fragment thereof, which binds to humanCD47, wherein said monoclonal antibody, or an antigen binding fragmentthereof, exhibits pH-dependent binding to CD47 present on a cell. 117.The monoclonal antibody, or an antigen binding fragment thereof, ofclaim 116, which: a. binds to human CD47; b. blocks SIRPα binding tohuman CD47; c. increases phagocytosis of human tumor cells; and d.induces death of human tumor cells.
 118. The monoclonal antibody, orantigen binding fragment thereof, of claim 116, wherein said monoclonalantibody, or an antigen binding fragment thereof, further comprises oneor more characteristics selected from the group consisting of: causes anincrease in cell surface calreticulin expression on human tumor cells;causes an increase in adenosine triphosphate (ATP) release by humantumor cells; causes an increase in high mobility group box 1 (HMGB1)release by human tumor cells; causes an increase in annexin A1 releaseby human tumor cells; causes an increase in Type I Interferon release byhuman tumor cells; causes an increase in C-X-C Motif Chemokine Ligand 10(CXCL10) release by human tumor cells; causes an increase in cellsurface protein disulfide-isomerase A3 (PDIA3) expression on human tumorcells; causes an increase in cell surface heat shock protein 70 (HSP70)expression on human tumor cells; and causes an increase in cell surfaceheat shock protein 90 (HSP90) expression on human tumor cells.
 119. Themonoclonal antibody, or antigen binding fragment thereof, of claim 116,wherein the antibody, or antigen binding fragment thereof, is a chimericor humanized antibody; or wherein the monoclonal antibody, or antigenbinding fragment thereof cross-reacts with one or more species homologsof CD47.
 120. The monoclonal antibody, or antigen binding fragmentthereof, of claim 116, wherein the pH-dependent binding to CD47 has agreater affinity for CD47 at an acidic pH compared to physiological pH.121. A method of treating a disease comprising an autoimmune disease, anautoinflammatory disease, an inflammatory disease, a cardiovasculardisease, or a cancer in a subject, comprising administration of themonoclonal antibody or antigen-binding fragment thereof, of claim 116,wherein the subject comprises a human or companion/pet animal, a workinganimal, a sport animal, a zoo animal, or a valuable animal kept incaptivity.
 122. The method of claim 121, wherein the disease is selectedfrom the group consisting of a leukemia, a lymphoma, ovarian cancer,breast cancer, endometrial cancer, colon cancer (colorectal cancer),rectal cancer, bladder cancer, urothelial cancer, lung cancer (non-smallcell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma ofthe lung), bronchial cancer, bone cancer, prostate cancer, pancreaticcancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer,bile duct cancer, esophageal cancer, renal cell carcinoma, thyroidcancer, squamous cell carcinoma of the head and neck (head and neckcancer), testicular cancer, cancer of the endocrine gland, cancer of theadrenal gland, cancer of the pituitary gland, cancer of the skin, cancerof soft tissues, cancer of blood vessels, cancer of brain, cancer ofnerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancerof hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma,meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma,neuroblastoma, melanoma, myelodysplastic syndrome, a sarcoma,ischemia-reperfusion injury, heart failure, arthritis, rheumatoidarthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, lupus, systemiclupus erythematous, juvenile rheumatoid arthritis, juvenile idiopathicarthritis, Grave's disease, Hashimoto's thyroiditis, Addison's disease,celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis,pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,uveitis, atherosclerosis, and ankylosing spondylitis.
 123. Themonoclonal antibody, or antigen binding fragment thereof, of claim 116,wherein the monoclonal antibody, or antigen binding fragment thereof,further exhibits reduced binding to a normal cell.
 124. A monoclonalantibody, or an antigen binding fragment thereof, which binds to humanCD47, wherein said monoclonal antibody, or an antigen binding fragmentthereof, exhibits reduced binding to a normal cell.
 125. The monoclonalantibody, or an antigen binding fragment thereof, of claim 124, which:a. binds to human CD47; b. blocks SIRPα binding to human CD47; c.increases phagocytosis of human tumor cells; and d. induces death ofhuman tumor cells.
 126. The monoclonal antibody, or antigen bindingfragment thereof, of claim 124, wherein the normal cell is selected fromthe group consisting of an endothelial cell, a skeletal muscle cell, anepithelial cell, a PBMC, a T cell, a red blood cell, a peripheral bloodmononuclear cell, a human aortic endothelial cell, a human skeletalmuscle cell, a human microvascular endothelial cell, a human renaltubular epithelial cell, a human peripherial blood CD3+ cell, and ahuman peripheral blood mononuclear cell.
 127. The monoclonal antibody,or antigen binding fragment thereof, of claim 124, wherein saidmonoclonal antibody, or an antigen binding fragment thereof, furthercomprises one or more characteristics selected from the group consistingof: causes an increase in cell surface calreticulin expression on humantumor cells; causes an increase in adenosine triphosphate (ATP) releaseby human tumor cells; causes an increase in high mobility group box 1(HMGB1) release by human tumor cells; causes an increase in annexin A1release by human tumor cells; causes an increase in Type I Interferonrelease by human tumor cells; causes an increase in C-X-C MotifChemokine Ligand 10 (CXCL10) release by human tumor cells; causes anincrease in cell surface protein disulfide-isomerase A3 (PDIA3)expression on human tumor cells; causes an increase in cell surface heatshock protein 70 (HSP70) expression on human tumor cells; and causes anincrease in cell surface heat shock protein 90 (HSP90) expression onhuman tumor cells.
 128. The monoclonal antibody, or antigen bindingfragment thereof, of claim 124, wherein the antibody, or antigen bindingfragment thereof, is a chimeric or humanized antibody; or wherein themonoclonal antibody, or antigen binding fragment thereof cross-reactswith one or more species homologs of CD47.
 129. A method of treating adisease comprising an autoimmune disease, an autoinflammatory disease,an inflammatory disease, a cardiovascular disease, or a cancer in asubject, comprising administration of the monoclonal antibody orantigen-binding fragment thereof, of claim 124, wherein the subjectcomprises a human or companion/pet animal, a working animal, a sportanimal, a zoo animal, or a valuable animal kept in captivity.
 130. Themethod of claim 129, wherein the disease is selected from the groupconsisting of a leukemia, a lymphoma, ovarian cancer, breast cancer,endometrial cancer, colon cancer (colorectal cancer), rectal cancer,bladder cancer, urothelial cancer, lung cancer (non-small cell lungcancer, adenocarcinoma of the lung, squamous cell carcinoma of thelung), bronchial cancer, bone cancer, prostate cancer, pancreaticcancer, gastric cancer, hepatocellular carcinoma, gall bladder cancer,bile duct cancer, esophageal cancer, renal cell carcinoma, thyroidcancer, squamous cell carcinoma of the head and neck (head and neckcancer), testicular cancer, cancer of the endocrine gland, cancer of theadrenal gland, cancer of the pituitary gland, cancer of the skin, cancerof soft tissues, cancer of blood vessels, cancer of brain, cancer ofnerves, cancer of eyes, cancer of meninges, cancer of oropharynx, cancerof hypopharynx, cancer of cervix, and cancer of uterus, glioblastoma,meduloblastoma, astrocytoma, glioma, meningioma, gastrinoma,neuroblastoma, melanoma, myelodysplastic syndrome, a sarcoma,ischemia-reperfusion injury, heart failure, arthritis, rheumatoidarthritis, multiple sclerosis, psoriasis, psoriatic arthritis, Crohn'sdisease, inflammatory bowel disease, ulcerative colitis, lupus, systemiclupus erythematous, juvenile rheumatoid arthritis, juvenile idiopathicarthritis, Grave's disease, Hashimoto's thyroiditis, Addison's disease,celiac disease, dermatomyositis, multiple sclerosis, myasthenia gravis,pernicious anemia, Sjogren syndrome, type I diabetes, vasculitis,uveitis, atherosclerosis, and ankylosing spondylitis.
 131. Themonoclonal antibody, or antigen binding fragment thereof, of claim 124,wherein the monoclonal antibody, or antigen binding fragment thereof,further exhibits pH-dependent binding to CD47 present on a cell.